Methods and compositions for regulating T cell subsets by modulating transcription factor activity

ABSTRACT

Methods for modulating production of a T helper type 2 (Th2)-associated cytokine, in particular interleukin-4, by modulating the activity of a transcription factor, in particular the proto-oncoprotein c-Maf, that regulates expression of the Th2-associated cytokine gene are disclosed. Methods for modulating development of T helper type 1 (Th1) or T helper type 2 (Th2) subsets in a subject using agents that modulate transcription factor activity are also disclosed. The methods of the invention can further involve use of agents that modulate the activity of additional transcription factors that contribute to the regulation of Th1- or Th2-associated cytokines, such as a Nuclear Factor of Activated T cells (NF-AT) protein and/or an AP-1 family protein. Compositions for modulating Th2-associated cytokine production, recombinant expression vectors and host cells, as well as screening assays to identify agents that modulate c-Maf activity, are also disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. Ser. No.08/636,602, filed on Apr. 23, 1996 now U.S. Pat. No. 5,958,671. Thecontents of that application are specifically incorporated herein byreference.

GOVERNMENT FUNDING

Work described herein was supported, at least in part, under grantAI37833 awarded by the National Institutes of Health. The U.S.government therefore may have certain rights in this invention.

BACKGROUND OF THE INVENTION

CD4+ T helper cells are not a homogeneous population but can be dividedon the basis of cytokine secretion into at least two subsets termed Thelper type 1 (Th1) and T helper type 2 (Th2) (see e.g., Mosmann, T. R.et al. (1986) J. Immunol. 136:2348-2357; Paul, W. E. and Seder, R. A.(1994) Cell 76:241-25; Seder, R. A. and Paul, W. E. (1994) Ann. Rev.Immunol. 12:635-673). Th1 cells secrete interleukin-2 (IL-2) andinterferon-γ (IFN-γ) while Th2 cells produce interleukin-4 (IL-4),interleukin-5 (IL-5), interleukin-10 (IL-10) and interleukin-13 (IL-13).Both subsets produce cytokines such as tumor necrosis factor (TNF) andgranulocyte/macrophage-colony stimulating factor (GM-CSF). In additionto their different pattern of cytokine expression, Th1 and Th2 cells arethought to have differing functional activities. For example, Th1 cellsare involved in inducing delayed type hypersensitivity responses,whereas Th2 cells are involved in providing efficient “help” to Blymphocytes and stimulating production of IgG1 and IgE antibodies.

There is now abundant evidence that the ratio of Th1 to Th2 cells ishighly relevant to the outcome of a wide array ofimmunologically-mediated clinical diseases including autoimmune,allergic and infectious diseases. For example, in experimentalleishmania infections in mice, animals that are resistant to infectionmount predominantly a Th1 response, whereas animals that are susceptibleto progressive infection mount predominantly a Th2 response (Heinzel, F.P., et al. (1989) J. Exp. Med. 169:59-72; Locksley, R. M. and Scott, P.(1992) Immunoparasitology Today 1:A58-A61). In murine schistosomiasis, aTh1 to Th2 switch is observed coincident with the release of eggs intothe tissues by female parasites and is associated with a worsening ofthe disease condition (Pearce, E. J., et al. (1991) J. Exp. Med.173:159-166; Grzych, J-M., et al. (1991) J. Immunol. 141:1322-1327;Kullberg, M. C., et al. (1992) J. Immunol. 148:3264-3270). Many humandiseases, including chronic infections (such as with humanimmunodeficiency virus (HIV) and tuberculosis) and certain metastaticcarcinomas, also are characterized by a Th1 to Th2 switch (see e.g.,Shearer, G. M. and Clerici, M. (1992) Prog. Chem. Immunol. 54:21-43;Clerici, M and Shearer, G. M. (1993) Immunology Today 14:107-111;Yamamura, M., et al. (1993) J. Clin. Invest. 91:1005-1010; Pisa, P., etal. (1992) Proc. Natl. Acad. Sci. USA 89:7708-7712; Fauci, A. S. (1988)Science 239:617-623). Furthermore, certain autoimmune diseases have beenshown to be associated with a predominant Th1 response. For example,patients with rheumatoid arthritis have predominantly Th1 cells insynovial tissue (Simon, A. K., et al. (1994) Proc. Natl. Acad. Sci. USA91:8562-8566) and experimental autoimmune encephalomyelitis (EAE) can beinduced by autoreactive Th1 cells (Kuchroo, V. K., et al. (1993) J.Immunol. 151:4371-4381).

The ability to alter or manipulate ratios of Th1 and Th2 subsetsrequires an understanding of the mechanisms by which the differentiationof CD4 T helper precursor cells (Thp), which secrete only IL-2, chooseto become Th1 or Th2 effector cells. It is clear that the cytokinesthemselves are potent Th cell inducers and form an autoregulatory loop(see e.g., Paul, W. E. and Seder, R. A. (1994) Cell 76:241-251; Seder,R. A. and Paul, W. E. (1994) Ann. Rev. Immunol. 12:635-673). Thus, IL-4promotes the differentiation of Th2 cells while preventing thedifferentiation of precursors into Th1 cells, while IL-12 and IFN-γ havethe opposite effect. One possible means therefore to alter Th1:Th2ratios is to increase or decrease the level of selected cytokines.Direct administration of cytokines or antibodies to cytokines has beenshown to have an effect on certain diseases mediated by either Th1 orTh2 cells. For example, administration of recombinant IL-4 or antibodiesto IL-12 ameliorate EAE, a Th1-driven autoimmune disease (see Racke; M.K. et al. (1994) J. Exp. Med. 180:1961-1966; and Leonard, J. P. et al.(1995) J. Exp. Med. 181:381-386), while anti-IL-4 antibodies cure theTh2-mediated parasitic disease, Leishmania major (Sadick, M. D. et al.(1990) J. Exp. Med. 171:115-127). However, as therapeutic options,systemic administration of cytokines or antibodies may have unwantedside effects and, accordingly, alternative approaches to manipulatingTh1/Th2 subsets are still needed.

The molecular basis for the tissue-specific expression of IL-4 in Th2cells, or any T cell cytokine, has remained elusive. One possibility isthe presence of repressor proteins that selectively silence cytokines.Transcriptional silencing has been well documented for bacteria, yeastand mammalian genes. Examples include E. coli thermoregulation genes(Goransson, M. et al. (1990) Nature 344:682-685), yeast α2 mating typegenes (Keleher, C. A. et al. (1988) Cell 53:927-936) and mammalian MHCclass I and TcRα genes (Weisman, J. D. and Singer, D. S. (1991) Mol.Cell. Biol. 11:4228-4234; Winoto, A. and Baltimore, D. (1989) Cell59:649-655). Indeed, early experiments involving injection of IL-2genomic DNA into Xenopus oocytes suggested the existence of a repressorprotein for IL-2 in resting versus activated T cell extracts (Mouzaki,A. et al. (1991) EMBO J. 10:1399-1406). These studies suggested that theabsence of IL-2 production in resting T cells was due to proteins thatsilenced the transcription of IL-2 by interacting with negative elementsin the IL-2 promoter.

A second possibility is the existence of Th selective transactivators. Afamily of four related transcription factors called Nuclear Factor ofActivated T cells (NF-AT), plays a key role in the regulation ofcytokine gene expression (see e.g., Emmel, E. A. et al. (1989) Science246:1617-1620; Flanagan, W. M. et al. (1991) Nature 352:803-807; Jain,J. et al. (1993) Nature 365:352-355; McCaffrey, P. G. et al. (1993)Science 262:750-754; Rao, A. (1994) Immunol. Today 15:274-281; Northrop,J. P. et al. (1994) Nature 369:497). However, NF-AT family members canbind to and transactivate the promoters of multiple cytokine genesincluding IL-2 and IL-4 (Rooney, J. et al. (1995) Immunity 2:545-553;Szabo, S. J. et al. (1993) Mol. Cell. Biol. 13:4793-4805; Flanagan, W.M. et al. (1991) Nature 352:803-807; Northrop, J. P. et al. (1994)Nature 369:497). Thus, they are not likely to be responsible fordirecting Th1- or Th2-specific cytokine transcription. Most, if not all,NF-AT binding sites in cytokine promoter regulatory regions areaccompanied by nearby sites that bind auxiliary transcription factors,usually members of the AP-1 family. It has been shown that NF-AT andAP-1 proteins bind coordinately and cooperatively and are required forfull activity of the IL-2 and IL-4 promoters. Different AP-1 proteins,specifically c-Jun, c-Fos, Fra-1, Fra-2, Jun B and Jun D, have beenshown to bind to these sites (Rao, A. et al. (1994) Immunol. Today15:274-281; Jain, J. et al. (1993) Nature 365:352-355; Boise, L. H. etal. (1993) Mol. Cell. Biol. 13:1911-1919; Rooney, J. et al. (1995)Immunity 2:545-553; Rooney, J. et al. (1995) Mol. Cell. Biol.15:6299-6310). However, none of these AP-1 proteins is expressed in aTh1- or Th2-specific manner and there is no evidence for thedifferential recruitment of AP-1 family members to the IL-2 or IL-4composite sites (Rooney, J. et al. (1995) Mol. Cell. Biol.15:6299-6310). Thus, neither NF-AT proteins nor the AP-1 family membersc-Jun, c-Fos, Fra-1, Fra-2, Jun B and Jun D can account for thetissue-specific transcription of IL-4 in Th2 cells.

SUMMARY OF THE INVENTION

This invention pertains to methods for regulating Th1 or Th2 subsets bymodulating the activity of a transcription factor that regulatesexpression of a Th2-specific cytokine gene. As described further herein,it has now been discovered that the tissue-specific expression of IL-4in Th2 cells is not due to a repressor protein but rather to aTh2-specific transactivator protein. The proto-oncogene c-maf has nowbeen demonstrated to be responsible for the tissue-specific expressionof the Th2-associated cytokine interleukin-4. Moreover, ectopicexpression of c-maf in cells other than Th2 cells (e.g, Th1 cells, Bcells and non-lymphoid cells) leads to activation of the IL-4 promoterand, under appropriate conditions, production of endogenous IL-4.

Accordingly, in one aspect the invention provides a method formodulating production of a T helper type 2 (Th2)-associated cytokine bya cell. The method involves contacting the cell with an agent thatmodulates the activity of a transcription factor that regulatesexpression of a Th2-associated cytokine gene such that production of theTh2-associated cytokine by a cell is modulated. In particular, theagents of the invention act intracellularly to modulate the activity ofa transcription factor that regulates expression of a Th2-associatedcytokine gene. Preferably, the transcription factor is a member of themaf family. Most preferably, the transcription factor is c-Maf. TheTh2-associated cytokine modulated in the method is preferablyinterleukin-4. In one embodiment, production of the Th2-associatedcytokine (e.g., IL-4) is stimulated, for example in a cell that does notnormally express the cytokine (such as a Th1 cell or a B cell). Avariety of agents can be used to stimulate cytokine production,including a nucleic acid molecule encoding a maf family protein that isintroduced into and expressed in the cell and chemical agents thatenhance the expression or activity of an endogenous maf family proteinin the cell. In another embodiment, production of a Th2-associatedcytokine by a cell (e.g., a Th2 cell) is inhibited. A variety of agentscan be used to inhibit cytokine production, including antisense nucleicacid molecules that are complementary to a maf family gene,intracellular antibodies that bind maf family proteins (e.g., in thecell nucleus), inhibitory forms of maf family proteins (e.g., dominantnegative forms) and chemical agents that inhibit the expression oractivity of an endogenous maf family protein in the cell. Cytokineproduction by the cell can be modulated in vitro or in vivo. In oneembodiment, a cell is contacted with a modulating agent in vitro andthen is administered to a subject to thereby regulate the development ofTh1 and/or Th2 subsets in the subject.

In another aspect, the invention provides methods for regulating thedevelopment of Th1 or Th2 subsets in a subject. In addition to theembodiment discussed above wherein ex vivo modified cells areadministered to the subject, in another embodiment, these methodsinvolve direct administration to the subject of an agent that modulatesthe activity of a transcription factor (e.g., a maf family member) thatregulates expression of a Th2-associated cytokine gene (e.g., IL-4) suchthat development of Th1 or Th2 cells in the subject is modulated.

The methods of the invention can further involve the use of additionalagents that modulate the activity of additional transcription factorsthat contribute to regulating the expression of Th1- or Th2-associatedcytokines. Preferred additional agents are those which modulate theactivity of a Nuclear Factor of Activated T cells (NF-AT) protein. Thus,in one embodiment, a stimulatory method of the invention can involve theuse of a first agent that stimulates the expression and/or activity of amaf protein and a second agent that stimulates the expression and/oractivity of an NF-AT protein. Similarly, an inhibitory method of theinvention can involve the use of a first agent that inhibits theexpression and/or activity of a maf protein and a second agent thatinhibits the expression and/or activity of an NF-AT protein.Alternatively or additionally, the modulatory methods of the inventioncan involve the use of additional agents that modulate the activity ofan AP-1 family protein.

The modulatory methods of the invention can be used to manipulateTh1:Th2 ratios in a variety of clinical situations. For example,inhibition of Th2 formation may be useful in allergic diseases,malignancies and infectious diseases whereas enhancement of Th2formation may be useful in autoimmune diseases and organtransplantation.

Another aspect of the invention pertains to compositions that are usefulfor modulating the production of a Th2-associated cytokine by a celland/or for modulating the development of Th1 or Th2 subsets in asubject. In one embodiment, these compositions include recombinantexpression vectors that encode a maf family protein, wherein themaf-encoding sequences are operatively linked to regulatory sequencesthat direct expression of the maf family protein in a specific celltype, such as lymphoid cells (e.g., T cells or B cells) or hematopoieticstem cells. In another embodiment, these compositions include hostcells, such as host lymphoid cells (e.g., host T cells or host B cells)or host hematopoietic stem cells, into which a recombinant expressionvector encoding a maf family protein has been introduced.

Yet another aspect of the invention pertains to screening assays foridentifying compounds that modulate the activity of a transcriptionfactor that regulates expression of a Th2-associated cytokine gene. Inone type of screening assay, an indicator cell which contains both 1) arecombinant expression vector encoding a transcription factor thatregulates expression of a Th2-associated cytokine gene and 2) a vectorcomprising regulatory sequences of the Th2-associated cytokine geneoperatively linked a reporter gene is used to identify compounds thatmodulate the expression and/or activity of the transcription factor. Inanother embodiment, a screening assay of the invention identifiesproteins from Th2 cells that form a protein-protein interaction with atranscription factor (e.g., c-Maf) that regulates expression of aTh2-associated cytokine gene. In yet another embodiment, a screeningassay of the invention identifies compounds that modulate theinteraction of c-Maf with a maf response element (MARE) in the promoterof a Th2-associated cytokine gene (e.g., a MARE in the IL-4 promoter).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a schematic of the cell fusion approach used to demonstratethat cytokine expression is not due to a repressor.

FIG. 1B is a reverse transcriptase-polymerase chain reaction (RT-PCR)analysis of Il-2 and IL-4 cytokine, and control β-actin, mRNA expressedby an unfused Th1 clone (D1.1), an unfused Th2 clone (D10), Th1 and Th2homokaryons and Th1-Th2 heterokaryons.

FIG. 2A is a Northern blot analysis depicting expression of an isolatedcDNA clone in Th1 cells, Th2 cells or B lymphoma cells. A control probespecific for GAPDH was used to show equal loading of RNA.

FIG. 2B is a Northern blot analysis depicting upregulated expression ofthe isolated cDNA clone during in vitro differentiation of normal naivespleen cells into Th2 cells. Total RNA was isolated from cells harvestedat the indicated time points. Culture supernatant at the appropriatedilution was measured for cytokine (IL-10) production by ELISA todetermine differentiation into the Th1 or Th2 lineage.

FIG. 3A is a bar graph depicting transactivation of the IL-4 promoter byc-Maf in a Th1 clone (AE7). AE7 cells were cotransfected with awild-type IL-4 CAT reporter construct and either a control plasmid(pMEX-NeoI), a c-Maf expression plasmid (pMEX-Maf) or a c-Fos expressionplasmid (pMEX-c-Fos). Half of each sample was stimulated 24 hours aftertransfection with antibodies to CD3. All samples were harvested 48 hoursafter transfection and relative CAT activities were determined.

FIG. 3B is a photograph of a thin layer chromatography plate depictingthe relative CAT activity in M12 B lymphoma cells cotransfected with awild-type IL-4 CAT reporter construct and either two control plasmids(pMEX-NeoI and pREP4), a c-Maf expression plasmid and a control plasmid(pMEX-Maf and pREP4), a c-Fos expression plasmid and a control plasmid(pMEX-c-Fos and pREP4), a c-Jun expression plasmid and a control plasmid(pMEX-c-Jun and pREP4), a control plasmid and an NF-ATp expressionplasmid (PMEX-NeoI and pREP-NFATp), a c-Maf expression plasmid and anNFATp expression plasmid (pMEX-Maf and pREP-NFATp) or a c-Fos expressionplasmid and an NF-ATp expression plasmid (pMEX-c-Fos and pREP-NFATp).Half of each sample was stimulated 24 hours after transfection with PMAand ionomycin. All samples were harvested 48 hours after transfectionand relative CAT activities were determined.

FIG. 4 is a bar graph depicting endogenous production of IL-4 in M12cells by ectopic expression of c-Maf and NF-ATp. Cells stablytransfected with the indicated control or expression plasmids wereeither unstimulated or stimulated with PMA and ionomycin for 24 hours.200 μl of supernatant from each sample was subjected to ELISA forcytokine quantitation.

FIG. 5A is a photograph of a DNAse I footprint gel of the IL-4 promoterperformed using nuclear extracts from Th2 (D10, CDC35) or Th1 (AE7, S53)clones harvested at the indicated time points after stimulation withanti-CD3 antibodies, which depicts a Th2-specific footprint immediatelydownstream of the putative MARE site in the IL-4 promoter. TwoTh2-specific hypersensitive residues on the non-coding strand of theIL-4 promoter are indicated by *. Five lanes of a DNAse I digestion ofthe IL-4 promoter probe (without nuclear extract) and a Maxam-GilbertA/G ladder were run next to the DNAse I treated samples.

FIG. 5B is a schematic representation of the proximal regulatory regionof the murine 4 promoter. The top portion shows the primary sequence ofthe murine IL-4 promoter (SEQ ID NO:5). The numbers indicated arerelative to the start site of transcription at +1. Asterisks denote theTh2-specific hypersensitive residues seen on DNAse I footprint. Thebottom portion shows the sequence of the wild type (−59 to −27)oligonucleotide (SEQ ID NO:1 and complement) and the 4 bp mutants (SEQID NO:6 through SEQ ID NO:12) used in EMSA and the functional assaysshown in FIGS. 6 and 7. Altered nucleotides are shown in lowercase boldand correspond to the numbering system shown in the top portion.

FIG. 6 is a photograph of an electrophoretic mobility shift assay (EMSA)demonstrating that c-Maf but not c-Jun binds to the proximal IL-4promoter and forms a complex with NF-ATp. EMSA was performed using theindicated proteins and labeled double-stranded oligonucleotides.

FIG. 7A is a bar graph (top) and a photograph of a thin layerchromatography plate (bottom) depicting the relative CAT activity in M12cells co-transfected with a c-Maf expression vector and either thewild-type IL-4 CAT reporter construct or one of the 4 bp mutants shownin FIG. 5B, demonstrating that transactivation of the IL-4 promoter byc-Maf maps to the MARE and Th2-specific footprint. The average of threeindependent experiments and one representative experiment are shown inthe top and bottom portions, respectively.

FIG. 7B is a photograph of an EMSA, performed using recombinant c-Maf,the IL-4 promoter (−59 to −27) probe and the indicated unlabeleddouble-stranded oligonucleotides as competitors, demonstrating thatbinding of recombinant c-Maf to the IL-4 promoter maps to the MARE andTh2-specific footprint.

DETAILED DESCRIPTION OF THE INVENTION

This invention pertains to methods and compositions for regulating Tcell subsets by modulating transcription factor activity, as well as toscreening assays for identifying compounds that can modulate suchtranscription factor activity. The invention is based, at least in part,on the discovery that Th2-specific expression of the interleukin-4 genedoes not result from the action of a specific repressor protein (asshown in Example 1) but rather from the action of a specifictransactivator protein. As described further herein, the transcriptionfactor responsible for Th2-specific expression of the interleukin-4 genehas now been identified as the c-Maf proto-oncoprotein, which isselectively expressed in differentiating and mature Th2 cells and absentfrom Th1 cells (see Example 2). Ectopic expression of c-Maf in cellsthat do not normally express it (such as Th1 cells and B cells) leads totransactivation of the IL-4 promoter (see Example 3) and, underappropriate conditions, to production of endogenous IL-4 (see Example4). Moreover, a protein present in nuclear extracts of Th2 cells, butnot Th1 cells, footprints the IL-4 promoter in the region of a mafresponse element (MARE) (see Example 5) and recombinant c-Maf binds tothe IL-4 promoter in vitro (see Example 6). The ability of c-Maf totransactivate IL-4 maps to the MARE and Th2-specific footprint in theIL-4 promoter (see Example 7).

The maf family of proteins is a sub-family of AP-1/CREB/ATF proteins.The v-maf oncogene was originally isolated from a spontaneousmusculoaponeurotic fibrosarcoma of chicken and identified as thetransforming gene of the avian retrovirus, AS42 (Nishizawa, M. et al.(1989) Proc. Natl. Acad. Sci. USA 86:7711-7715). V-maf encodes a 42 kdbasic region/leucine zipper (b-zip) protein with homology to the c-fosand c-jun oncogenes. Its cellular homologue, the c-maf proto-oncogenehas only two structural changes in the coding region from v-maf(Kataoka, K. et al. (1993) J. Virol. 67:2133-2141). The maf familyincludes c-maf, mafB, a human retina-specific gene Nrl (Swaroop, A. etal. (1992) Proc. Natl. Acad. Sci. USA 89:266-270), mafK, mafF, mafG andp18. The latter four, mafK, mafF, mafG and p18, each encode proteinsthat lack the amino terminal two thirds of c-Maf that contains thetransactivating domain (Fujiwara, K. T. et al. (1993) Oncogene8:2371-2380; Igarashi, K. et al. (1995) J. Biol. Chem. 270:7615-7624;Andrews, N. C. et al. (1993) Proc. Natl. Acad. Sci. USA 90:11488-11492;Kataoka, K. et al. (1995) Mol. Cell. Biol. 15:2180-2190) and arereferred to herein as “small” mafs. C-maf and maf family members formhomodimers and heterodimers with each other and with Fos and Jun,consistent with the known ability of the AP-1 proteins to pair with eachother (Kerppola, T. K. and Curran, T. (1994) Oncogene 9:675-684;Kataoka, K. et al. (1994) Mol. Cell. Biol. 14:700-712). The DNA targetsequence to which c-Maf homodimers bind, termed the c-Maf responseelement (MARE), is a 13 or 14 bp element which contains a core TRE(T-MARE) or CRE (C-MARE) palindrome respectively. Prior to the presentinvention, little was known about the function of maf family members,although c-Maf has been shown to stimulate transcription from thePurkinje neuron-specific promoter L7 (Kurscher, C. and Morgan, J. I.(1994) Mol. Cell. Biol. 15:246-254) and Nrl has been shown to driveexpression of the QR1 retina-specific gene (Swaroop, A. et al. (1992)Proc. Natl. Acad. Sci. USA 89:266-270). The small mafs have been shownto function as repressors of α and β-globin transcription when bound ashomodimers but are essential as heterodimeric partners with theerythroid-specific factor p45NF-E2 to activate globin gene transcription(Kataoka, K. et al. (1995) Mol. Cell. Biol. 15:2180-2190; Igarashi, K.et al. (1994) Nature 367:568-572). MafK overexpression has been shown toinduce erythroleukemia cell differentiation (Igarashi, K. et al. (1995)Proc. Natl. Acad Sci. USA 92:7445-7449). However, prior to the presentinvention, there have been no reports implicating c-maf or maf familymembers in the regulation of genes expressed in lymphoid cells or incytokine gene expression in any tissue.

Various aspects of the present invention are described in further detailin the following subsections.

I. Modulation of Th2-Associated Cytokine Production

As discussed above, the transcription factor responsible for theTh2-specific expression of the interleukin-4 gene has now beenidentified as the c-Maf proto-oncogene. Modulation of the expressionand/or activity of c-Maf, therefore, provides a means to regulate theproduction of interleukin-4. Since IL-4 itself serves an autoregulatoryfunction in the development of Th2 cells (see e.g., Paul, W. E. andSeder, R. A. (1994) Cell 76:241-251; Seder, R. A. and Paul, W. E. (1994)Ann. Rev. Immunol. 12:635-673), and thus production of IL-4 can lead tothe production of additional Th2-associated cytokines such as IL-5,IL-10 and IL-13 through further Th2 differentiation, modulation of c-Mafexpression and/or activity provides a general approach for modulatingproduction of Th2-associated cytokines.

Accordingly, the invention provides a method for modulating productionof a Th2-associated cytokine by a cell. This method involves contactingthe cell with an agent that modulates the activity of a transcriptionfactor that regulates expression of a Th2-associated cytokine gene suchthat production of the Th2-associated cytokine by a cell is modulated.In particular, the modulatory agents of the invention are characterizedby acting intracellularly to modulate the activity of a transcriptionfactor. Preferably, the transcription factor is a member of the maffamily and most preferably is c-Maf. As used herein, the term“Th2-associated cytokine” is intended to refer to a cytokine that isproduced preferentially or exclusively by Th2 cells rather than by Th1cells. Preferably the Th2-associated cytokine is interleukin-4. As usedherein, the term “contacting” (i.e., contacting a cell with an agent) isintended to include incubating the agent and the cell together in vitro(e.g., adding the agent to cells in culture) and administering the agentto a subject such that the agent and cells of the subject are contactedin vivo. As used herein, the various forms of the term “modulation” areintended to include stimulation (e.g., increasing or upregulating aparticular response or activity) and inhibition (e.g., decreasing ordownregulating a particular response or activity). Accordingly, in oneembodiment of the method of the invention, production of aTh2-associated cytokine by the cell is stimulated by contacting the cellwith a stimulatory agent that stimulates c-Maf expression and/oractivity. In another embodiment of the method of the invention,production of a Th2-associated cytokine by the cell is inhibited bycontacting the cell with a inhibitory agent that inhibits c-Mafexpression and/or activity.

As demonstrated in the Examples, although c-Maf is responsible for thetissue specificity of IL-4 gene expression, c-Maf acts synergisticallywith one or more additional transcription factors to activate IL-4 genetranscription. In particular, c-Maf acts synergistically with an NF-ATprotein to stimulate IL-4 gene expression. Moreover, NF-AT proteins andother members of the AP-1/CREB/ATF family of transcription factors havebeen demonstrated to be involved in regulating expression of both Th1-and Th2-associated cytokine genes. Accordingly, the method of theinvention for modulating Th2-associated cytokine production by a cellcan further comprise contacting the cell with a second agent thatmodulates (i.e., stimulates or inhibits) the expression or activity of asecond transcription factor that contributes to regulating theexpression of a Th1- or Th2-associated cytokine gene (discussed furtherbelow).

A. Stimulatory Agents

According to the method of the invention, to stimulate Th2-associatedcytokine production by a cell, the cell is contacted with a stimulatoryagent that stimulates expression and/or activity of a transcriptionfactor (e.g., c-Maf) that regulates expression of a Th2-associatedcytokine gene. Th2-associated cytokine production can be stimulated incell types that do not normally express such cytokines, such as Th1cells, B cells or non-lymphoid cells. Furthermore, Th2-associatedcytokine production can be stimulated in helper precursor cells (Thp) topromote their differentiation along the Th2 pathway instead of the Th1pathway.

A preferred stimulatory agent is a nucleic acid molecule encoding a maffamily protein, wherein the nucleic acid molecule is introduced into thecell in a form suitable for expression of the maf family protein in thecell. For example, a c-Maf cDNA is cloned into a recombinant expressionvector and the vector is transfected into the cell. As demonstrated inExample 3, ectopic expression of a c-maf recombinant expression vectorin Th1 cells, B cells or non-lymphoid cells leads to activation of theIL-4 promoter. Additionally, under appropriate conditions (discussed infurther detail below), transcription of the endogenous IL-4 gene isstimulated, leading to IL-4 production by cells that do not normallyexpress this cytokine (see Example 4).

To express a maf family protein in a cell, typically a maf family cDNAis first introduced into a recombinant expression vector using standardmolecular biology techniques. A maf family cDNA can be obtained, forexample, by amplification using the polymerase chain reaction (PCR) orby screening an appropriate cDNA library. The nucleotide sequences ofmaf family cDNAs are known in the art and can be used for the design ofPCR primers that allow for amplification of a cDNA by standard PCRmethods or for the design of a hybridization probe that can be used toscreen a cDNA library using standard hybridization methods. Preferably,the maf family cDNA is that of the c-maf proto-oncogene. The nucleotideand predicted amino acid sequences of a mammalian (mouse) c-maf cDNA aredisclosed in Kurscher C. and Morgan, J. I. (1995) Mol. Cell. Biol.15:246-254 and deposited in the GenBank database at accession numberS74567. This mammalian c-maf is highly homologous to the avian v-mafsequence (disclosed in Nishizawa, M. K. et al. (1989) Proc. Natl. AcadSci. USA 86:7711-7715 and GenBank accession numbers D28598 and D28596),indicating that c-maf is well conserved among species. c-maf cDNAs fromother mammalian species, including humans, can be isolated usingstandard molecular biology techniques (e.g., PCR or cDNA libraryscreening) and primers or probes designed based upon the mouse or aviansequences. Human partial cDNA sequences homologous to the mouse c-mafcDNA are also deposited in the GenBank database at accession numbersH24189 and N75504. The sequences of other maf family members are alsoknown in the art, for example MafB (Kataoka, K. et al. (1994) Mol. CellBiol. 14:7581-91; GenBank accession number D28600), MafG (Kataoka et al.(1994) Mol. Cell Biol. 14:7581-91; GenBank accession numbers D28601 andD28602), MafF (GenBank accession number D16184) and MafK (Igarashi, K.et al. (1995) J. Biol. Chem. 270:7615-7624; GenBank accession numbersD16187 and D42124).

Following isolation or amplification of a maf family cDNA, the DNAfragment is introduced into an expression vector. As used herein, theterm “vector” refers to a nucleic acid molecule capable of transportinganother nucleic acid to which it has been linked. One type of vector isa “plasmid”, which refers to a circular double stranded DNA loop intowhich additional DNA segments may be ligated. Another type of vector isa viral vector, wherein additional DNA segments may be ligated into theviral genome. Certain vectors are capable of autonomous replication in ahost cell into which they are introduced (e.g., bacterial vectors havinga bacterial origin of replication and episomal mammalian vectors). Othervectors (e.g., non-episomal mammalian vectors) are integrated into thegenome of a host cell upon introduction into the host cell, and therebyare replicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “recombinantexpression vectors” or simply “expression vectors”. In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. In the present specification, “plasmid” and“vector” may be used interchangeably as the plasmid is the most commonlyused form of vector. However, the invention is intended to include suchother forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, adenoviruses and adeno-associatedviruses), which serve equivalent functions.

The recombinant expression vectors of the invention comprise a nucleicacid in a form suitable for expression of the nucleic acid in a hostcell, which means that the recombinant expression vectors include one ormore regulatory sequences, selected on the basis of the host cells to beused for expression and the level of expression desired, which isoperatively linked to the nucleic acid sequence to be expressed. Withina recombinant expression vector, “operably linked” is intended to meanthat the nucleotide sequence of interest is linked to the regulatorysequence(s) in a manner which allows for expression of the nucleotidesequence (e.g., in an in vitro transcription/translation system or in ahost cell when the vector is introduced into the host cell). The term“regulatory sequence” is intended to includes promoters, enhancers andother expression control elements (e.g., polyadenylation signals). Suchregulatory sequences are described, for example, in Goeddel; GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990). Regulatory sequences include those which directconstitutive expression of a nucleotide sequence in many types of hostcell, those which direct expression of the nucleotide sequence only incertain host cells (e.g., tissue-specific regulatory sequences) or thosewhich direct expression of the nucleotide sequence only under certainconditions (e.g., inducible regulatory sequences). It will beappreciated by those skilled in the art that the design of theexpression vector may depend on such factors as the choice of the hostcell to be transformed, the level of expression of protein desired, etc.When used in mammalian cells, the expression vector's control functionsare often provided by viral regulatory elements. For example, commonlyused promoters are derived from polyoma virus, adenovirus,cytomegalovirus and Simian Virus 40. Non-limiting examples of mammalianexpression vectors include pCDM8 (Seed, B., (1987) Nature 329:840) andpMT2PC (Kaufman et al. (1987), EMBO J. 6:187-195). A variety ofmammalian expression vectors carrying different regulatory sequences arecommercially available. For constitutive expression of the nucleic acidin a mammalian host cell, a preferred regulatory element is thecytomegalovirus promoter/enhancer. Moreover, inducible regulatorysystems for use in mammalian cells are known in the art, for examplesystems in which gene expression is regulated by heavy metal ions (seee.g., Mayo et al. (1982) Cell 29:99-108; Brinster et al. (1982) Nature296:39-42; Searle et al. (1985) Mol. Cell. Biol. 5:1480-1489), heatshock (see e.g., Nouer et al. (1991) in Heat Shock Response, e.d. Nouer,L. , CRC, Boca Raton, Fla., pp167-220), hormones (see e.g., Lee et al.(1981) Nature 294:228-232; Hynes et al. (1981) Proc. Natl. Acad Sci. USA78:2038-2042; Klock et al. (1987) Nature 329:734-736; Israel & Kaufman(1989) Nucl. Acids Res. 17:2589-2604; and PCT Publication No. WO93/23431), FK506-related molecules (see e.g., PCT Publication No. WO94/18317) or tetracyclines (Gossen, M. and Bujard, H. (1992) Proc. Natl.Acad. Sci. USA 89:5547-5551; Gossen, M. et al. (1995) Science268:1766-1769; PCT Publication No. WO 94/29442; and PCT Publication No.WO 96/01313). Still further, many tissue-specific regulatory sequencesare known in the art, including the albumin promoter (liver-specific;Pinkert et al. (1987) Genes Dev. 1:268-277), lymphoid-specific promoters(Calarne and Eaton (1988) Adv. Immunol. 43:235-275), in particularpromoters of T cell receptors (Winoto and Baltimore (1989) EMBO J.8:729-733) and immunoglobulins (Banerji et al. (1983) Cell 33:729-740;Queen and Baltimore (1983) Cell 33:741-748), neuron-specific promoters(e.g., the neurofilament promoter; Byrne and Ruddle (1989) Proc. Natl.Acad. Sci. USA 86:5473-5477), pancreas-specific promoters (Edlund et al.(1985) Science 230:912-916) and mammary gland-specific promoters (e.g.,milk whey promoter, U.S. Pat. No. 4,873,316 and European ApplicationPublication No. 264,166). Developmentally-regulated promoters are alsoencompassed, for example the murine hox promoters (Kessel and Gruss(1990) Science 249:374-379) and the α-fetoprotein promoter (Campes andTilghman (1989) Genes Dev. 3:537-546).

Vector DNA can be introduced into mammalian cells via conventionaltransfection techniques. As used herein, the various forms of the term“transfection” are intended to refer to a variety of art-recognizedtechniques for introducing foreign nucleic acid (e.g., DNA) intomammalian host cells, including calcium phosphate co-precipitation,DEAE-dextran-mediated transfection, lipofection, or electroporation.Suitable methods for transfecting host cells can be found in Sambrook etal. (Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold SpringHarbor Laboratory press (1989)), and other laboratory manuals.

For stable transfection of mammalian cells, it is known that, dependingupon the expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome. Inorder to identify and select these integrants, a gene that encodes aselectable marker (e.g., resistance to antibiotics) is generallyintroduced into the host cells along with the gene of interest.Preferred selectable markers include those which confer resistance todrugs, such as G418, hygromycin and methotrexate. Nucleic acid encodinga selectable marker may be introduced into a host cell on a separatevector from that encoding a maf family protein or, more preferably, onthe same vector. Cells stably transfected with the introduced nucleicacid can be identified by drug selection (e.g., cells that haveincorporated the selectable marker gene will survive, while the othercells die).

Another form of a stimulatory agent for stimulating expression of aTh2-associated cytokine in a cell is a chemical compound that stimulatesthe expression or activity of an endogenous maf family protein in thecell. Such compounds can be identified using screening assays thatselect for compounds that stimulate the expression or activity of a maffamily protein. Examples of suitable screening assays are described infurther detail in subsection V below.

In addition to use of an agent that stimulates the expression oractivity of a maf family protein, the stimulatory methods of theinvention can involve the use of a second agent that stimulates theexpression or activity of a second transcription factor that contributesto regulating the expression of a Th1- or Th2-associated cytokine gene.In Example 4, it is shown that stimulation of the expression ofendogenous IL-4 in M12 B lymphoma cells required the introduction intothe cells of both a c-Maf expression vector and an NF-AT expressionvector, thereby demonstrating that c-Maf and NF-AT act synergisticallyto activate IL-4 transcription, with c-maf responsible for thetissue-specificity of expression. While the skilled artisan willappreciate that certain cells may express sufficient amounts ofendogenous NF-AT such that use of a second agent that stimulates theexpression or activity of NF-AT is unnecessary, in certain situationsand with certain cell types it may be necessary M to stimulate bothc-Maf and NF-AT to achieve IL-4 production.

Accordingly, in one embodiment, the stimulatory methods of the inventioninvolve the use of a first agent that stimulates the expression oractivity of c-Maf and a second agent that stimulates the expression oractivity of an NF-AT protein. A preferred second agent for stimulatingNF-AT activity in a cell is a recombinant expression encoding an NF-AT,wherein the recombinant expression vector is introduced into the celland NF-AT is expressed in the cell. NF-AT-encoding expression vectorscan be prepared and introduced into cells as described above for c-Mafexpression vectors. The nucleotide sequences of NF-AT cDNAs are known inthe art and can be used for the design of PCR primers that allow foramplification of a cDNA by standard PCR methods or for the design of ahybridization probe that can be used to screen a cDNA library usingstandard hybridization methods. Four NF-AT family members have beenidentified (see e.g., Emmel, E. A. et al. (1989) Science 246:1617-1620;Flanagan, W. M. et al. (1991) Nature 352:803-807; Jain, J. et al. (1993)Nature 365:352-355; McCaffrey, P. G. et al. (1993) Science 262:750-754;Rao, A. (1994) Immunol. Today 15:274-281; Northrop, J. P. et al. (1994)Nature 369:497). Preferably, the NF-AT cDNA is that of NF-ATp. Thenucleotide and predicted amino acid sequences of a mammalian NF-ATp cDNAare disclosed in McCaffrey, P. G. et al. (1993) Science 262:750-754. Thenucleotide and predicted amino acid sequences of a mammalian NF-ATc cDNAare disclosed in Northrop, J. P. et al. (1994) Nature 369:497 anddeposited in the GenBank database at accession number U08015. Thenucleotide and predicted amino acid sequences of mammalian NF-AT3 andNF-AT4 cDNAs are disclosed in Hoey, T. et al. (1995) Immunity 2:461-472.

Alternative to use of an NF-AT cDNA to stimulate the activity of NF-ATin a cell, one or more chemical compounds that stimulate NF-AT activityin a cell can be used as a second agent in a stimulatory method of theinvention. Compounds that stimulate NF-AT activity in cells are known inthe art (for a review see Rao, A. (1994) Immunol. Today 15:274-281). Forexample, stimulation of certain cells with the phorbol ester phorbolmyristate acetate (PMA) and a calcium ionophore (e.g., ionomycin)results in translocation of NF-ATs to the cell nucleus (see e.g.,Flanagan, W. M. et al. (1991) Nature 352:803-807; Jain, J. et al. (1993)Nature 365:352-355). Additionally, stimulation of T cells through the Tcell receptor (TcR), for example with an anti-CD3 antibody, results inactivation of NF-AT in the T cells.

In addition to NF-AT proteins, AP-1 family members, including c-Jun,c-Fos, Fra-1, Fra-2, Jun B and Jun D, have been shown to be involved inregulating the expression of both Th1- and Th2-associated cytokine genes(e.g., IL-2 and IL-4) (see e.g., Rao, A. et al. (1994) Immunol. Today15:274-281; Jain, J. et al. (1993) Nature 365:352-355; Boise, L. H. etal. (1993) Mol. Cell. Biol. 13:1911-1919; Rooney, J. et al. (1995)Immunity 2:545-553; Rooney, J. et al. (1995) Mol. Cell. Biol.15:6299-6310). Although these factors are not responsible for theTh1/Th2 specificity of expression of the cytokine genes, and thesefactors do not appear to synergize with c-Maf in regulating IL-4 geneexpression (see the Examples), AP-1 family members have been shown toincrease IL-4 expression in Th2 cells (see e.g., Rooney, J. et al.(1995) Immunity 2:545-553). Accordingly, in certain circumstances it maybe beneficial, in addition to stimulating c-Maf activity (and possiblyNF-AT activity), also to stimulate the activity of an AP-1 protein.Accordingly, in one embodiment, the stimulatory methods of the inventioninvolve the use of a first agent that stimulates the expression oractivity of c-Maf and a second agent that stimulates the expression oractivity of an AP-1 protein. In another embodiment, the inventioninvolves the use of a first agent that stimulates the expression oractivity of c-Maf, a second agent that stimulates the expression oractivity of an NF-AT protein and a third agent that stimulates theexpression or activity of an AP-1 protein.

A preferred agent for stimulating AP-1 activity in a cell is arecombinant expression encoding an AP-1 protein, wherein the recombinantexpression vector is introduced into the cell and the AP-1 protein isexpressed in the cell. AP-1-encoding expression vectors can be preparedand introduced into cells as described above for c-Maf expressionvectors. The nucleotide sequences of AP-1 cDNAs are known in the art.For example, the nucleotide and predicted amino acid sequences of humanc-fos are disclosed in van Straaten, F. et al. (1983) Proc. Natl. Acad.Sci. USA 80:3183-3187. The nucleotide and predicted amino acid sequencesof human c-jun are disclosed in Bohmann, D. et al. (1987) Science238:1386-1392. The nucleotide and predicted amino acid sequences ofhuman jun-B and jun-D are disclosed in Nomura, N. et al. (1990) Nucl.Acids Res. 18:3047-3048. The nucleotide and predicted amino acidsequences of human fra-1 and fra-2 are disclosed in Matsui, M. et al.(1990) Oncogene 5:249-255. These sequences can be used for the design ofPCR primers that allow for amplification of a cDNA by standard PCRmethods or for the design of a hybridization probe that can be used toscreen a cDNA library using standard hybridization methods.Alternatively, one or more chemical compounds that stimulate AP-1activity in a cell can be used as additional agents in a stimulatorymethod of the invention. Compounds that stimulate AP-1 activity in cellsare known in the art, including PMA/calcium ionophore (e.g., ionomycin)and anti-CD3 antibodies.

B. Inhibitory Agents

According to the method of the invention, to inhibit Th2-associatedcytokine production by a cell, the cell is contacted with an inhibitoryagent that inhibits expression and/or activity of a transcription factor(e.g., c-Maf) that regulates expression of a Th2-associated cytokinegene. Th2-associated cytokine production can be inhibited in, forexample, Th2 cells or in helper precursor cells (Thp) to promote theirdifferentiation along the Th1 pathway instead of the Th2 pathway.Inhibitory agents of the invention can be, for example, intracellularbinding molecules that act to inhibit the expression or activity of thetranscription factor. As used herein, the term “intracellular bindingmolecule” is intended to include molecules that act intracellularly toinhibit the expression or activity of a protein by binding to theprotein or to a nucleic acid (e.g., an mRNA molecule) that encodes theprotein. Examples of intracellular binding molecules, described infurther detail below, include antisense nucleic acids, intracellularantibodies and dominant negative inhibitors.

In one embodiment, an inhibitory agent of the invention is an antisensenucleic acid molecule that is complementary to a gene encoding a maffamily protein, or to a portion of said gene, or a recombinantexpression vector encoding said antisense nucleic acid molecule. The useof antisense nucleic acids to downregulate the expression of aparticular protein in a cell is well known in the art (see e.g.,Weintraub, H. et al., Antisense RNA as a molecular tool for geneticanalysis, Reviews—Trends in Genetics, Vol. 1(1) 1986; Askari, F. K. andMcDonnell, W. M. (1996) N. Eng. J. Med. 3:316-318; Bennett, M. R. andSchwartz, S. M. (1995) Circulation 92:1981-1993; Mercola, D. and Cohen,J. S. (1995) Cancer Gene Ther. 2:47-59; Rossi, J. J. (1995) Br. Med.Bull. 51:217-225; Wagner, R. W. (1994) Nature 372:333-335). An antisensenucleic acid molecule comprises a nucleotide sequence that iscomplementary to the coding strand of another nucleic acid molecule(e.g., an mRNA sequence) and accordingly is capable of hydrogen bondingto the coding strand of the other nucleic acid molecule. Antisensesequences complementary to a sequence of an mRNA can be complementary toa sequence found in the coding region of the mRNA, the 5′ or 3′untranslated region of the mRNA or a region bridging the coding regionand an untranslated region (e.g., at the junction of the 5′ untranslatedregion and the coding region). Furthermore, an antisense nucleic acidcan be complementary in sequence to a regulatory region of the geneencoding the mRNA, for instance a transcription initiation sequence orregulatory element. Preferably, an antisense nucleic acid is designed soas to be complementary to a region preceding or spanning the initiationcodon on the coding strand or in the 3′ untranslated region of an mRNA.An antisense nucleic acid for inhibiting the expression of a Maf familyprotein in a cell can be designed based upon the nucleotide sequenceencoding the Maf family protein, constructed according to the rules ofWatson and Crick base pairing.

An antisense nucleic acid can exist in a variety of different forms. Forexample, the antisense nucleic acid can be an oligonucleotide that iscomplementary to only a portion of a maf family gene. An antisenseoligonucleotides can be constructed using chemical synthesis proceduresknown in the art. An antisense oligonucleotide can be chemicallysynthesized using naturally occurring nucleotides or variously modifiednucleotides designed to increase the biological stability of themolecules or to increase the physical stability of the duplex formedbetween the antisense and sense nucleic acids, e.g. phosphorothioatederivatives and acridine substituted nucleotides can be used. To inhibitMaf protein expression in cells in culture, one or more antisenseoligonucleotides can be added to cells in culture media, typically at200 μg oligonucleotide/ml.

Alternatively, an antisense nucleic acid can be produced biologicallyusing an expression vector into which a nucleic acid has been subclonedin an antisense orientation (i.e., nucleic acid transcribed from theinserted nucleic acid will be of an antisense orientation to a targetnucleic acid of interest). Regulatory sequences operatively linked to anucleic acid cloned in the antisense orientation can be chosen whichdirect the expression of the antisense RNA molecule in a cell ofinterest, for instance promoters and/or enhancers or other regulatorysequences can be chosen which direct constitutive, tissue specific orinducible expression of antisense RNA. The antisense expression vectoris prepared as described above for recombinant expression vectors,except that the cDNA (or portion thereof) is cloned into the vector inthe antisense orientation. The antisense expression vector can be in theform of, for example, a recombinant plasmid, phagemid or attenuatedvirus. The antisense expression vector is introduced into cells using astandard transfection technique, as described above for recombinantexpression vectors.

In another embodiment, an antisense nucleic acid for use as aninhibitory agent is a ribozyme. Ribozymes are catalytic RNA moleculeswith ribonuclease activity which are capable of cleaving asingle-stranded nucleic acid, such as an mRNA, to which they have acomplementary region (for reviews on ribozymes see e.g., Ohkawa, J. etal. (1995) J. Biochem. 118:251-258; Sigurdsson, S. T. and Eckstein, F.(1995) Trends Biotechnol. 13:286-289; Rossi, J. J. (1995) TrendsBiotechnol. 13:301-306; Kiehntopf, M. et al. (1995) J. Mol. Med.73:65-71). A ribozyme having specificity for a maf family mRNA can bedesigned based upon the nucleotide sequence of the maf family,preferably c-maf. For example, a derivative of a Tetrahymena L-19 IVSRNA can be constructed in which the base sequence of the active site iscomplementary to the base sequence to be cleaved in a c-maf mRNA. Seefor example U.S. Pat. Nos. 4,987,071 and 5,116,742, both by Cech et al.Alternatively, c-maf mRNA can be used to select a catalytic RNA having aspecific ribonuclease activity from a pool of RNA molecules. See forexample Bartel, D. and Szostak, J. W. (1993) Science 261: 1411-1418.

Another type of inhibitory agent that can be used to inhibit theexpression and/or activity of a Maf protein in a cell is anintracellular antibody specific for the Maf protein. The use ofintracellular antibodies to inhibit protein function in a cell is knownin the art (see e.g., Carlson, J. R. (1988) Mol. Cell. Biol.8:2638-2646; Biocca, S. et al. (1990) EMBO J. 9:101-108; Werge, T. M. etal. (1990) FEBS Letters 274:193-198; Carlson, J. R. (1993) Proc. Natl.Acad. Sci. USA 90:7427-7428; Marasco, W. A. et al. (1993) Proc. Natl.Acad. Sci. USA 90:7889-7893; Biocca, S. et al. (1994) Bio/Technology12:396-399; Chen, S-Y. et al. (1994) Human Gene Therapy 5:595-601; Duan,L. et al. (1994) Proc. Natl. Acad. Sci. USA 91:5075-5079; Chen, S-Y. etal. (1994) Proc. Natl. Acad. Sci. USA 91:5932-5936; Beerli, R. R. et al.(1994) J. Biol. Chem. 269:23931-23936; Beerli, R. R. et al. (1994)Biochem. Biophys. Res. Commun. 204:666-672; Mhashilkar, A. M. et al.(1995) EMBO J. 14:1542-1551; Richardson, J. H. et al. (1995) Proc. Natl.Acad Sci. USA 92:3137-3141; PCT Publication No. WO 94/02610 by Marascoet al.; and PCT Publication No. WO 95/03832 by Duan et al.).

To inhibit protein activity using an intracellular antibody, arecombinant expression vector is prepared which encodes the antibodychains in a form such that, upon introduction of the vector into a cell,the antibody chains are expressed as a functional antibody in anintracellular compartment of the cell. For inhibition of transcriptionfactor activity according to the inhibitory methods of the invention,preferably an intracellular antibody that specifically binds thetranscription factor is expressed within the nucleus of the cell.Nuclear expression of an intracellular antibody can be accomplished byremoving from the antibody light and heavy chain genes those nucleotidesequences that encode the N-terminal hydrophobic leader sequences andadding nucleotide sequences encoding a nuclear localization signal ateither the N- or C-terminus of the light and heavy chain genes (seee.g., Biocca, S. et al. (1990) EMBO J. 9:101-108; Mhashilkar, A. M. etal. (1995) EMBO J. 14:1542-1551). A preferred nuclear localizationsignal to be used for nuclear targeting of the intracellular antibodychains is the nuclear localization signal of SV40 Large T antigen (seeBiocca, S. et al. (1990) EMBO J. 9:101-108; Mhashilkar, A. M. et al.(1995) EMBO J. 14:1542-1551).

To prepare an intracellular antibody expression vector, antibody lightand heavy chain cDNAs encoding antibody chains specific for the targetprotein of interest, e.g., a Maf family protein, are isolated, typicallyfrom a hybridoma-that secretes a monoclonal antibody specific for themaf protein. Preparation of antisera against Maf family proteins hasbeen described in the art (see e.g., Fujiwara, K. T. et al. (1993)Oncogene 8:2371-2380; Kataoka, K. et al. (1993) J. Virol. 67:2133-2141;Kerppola, T. K. and Curran, T. (1994) Oncogene 9:675-684; Igarashi, K.et al. (1995) Proc. Natl. Acad. Sci. USA 92:7445-7449). Anti-Maf proteinantibodies can be prepared by immunizing a suitable subject, (e.g.,rabbit, goat, mouse or other mammal) with a Maf protein immunogen. Anappropriate immunogenic preparation can contain, for examples,recombinantly expressed Maf protein or a chemically synthesized Mafpeptide. The preparation can further include an adjuvant, such asFreund's complete or incomplete adjuvant, or similar immunostimulatoryagent. Antibody-producing cells can be obtained from the subject andused to prepare monoclonal antibodies by standard techniques, such asthe hybridoma technique originally described by Kohler and Milstein(1975, Nature 256:495-497) (see also, Brown et al. (1981) J. Immunol127:539-46; Brown et al. (1980) J Biol Chem 255:4980-83; Yeh et al.(1976) PNAS 76:2927-31; and Yeh et al. (1982) Int. J. Cancer 29:269-75).The technology for producing monoclonal antibody hybridomas is wellknown (see generally R. H. Kenneth, in Monoclonal Antibodies: A NewDimension In Biological Analyses, Plenum Publishing Corp., New York,N.Y. (1980); E. A. Lerner (1981) Yale J. Biol. Med., 54:387-402; M. L.Gefter et al. (1977) Somatic Cell Genet., 3:231-36). Briefly, animmortal cell line (typically a myeloma) is fused to lymphocytes(typically splenocytes) from a mammal immunized with a maf proteinimmunogen as described above, and the culture supernatants of theresulting hybridoma cells are screened to identify a hybridoma producinga monoclonal antibody that binds specifically to the Maf protein. Any ofthe many well known protocols used for fusing lymphocytes andimmortalized cell lines can be applied for the purpose of generating ananti-Maf protein monoclonal antibody (see, e.g., G. Galfre et al. (1977)Nature 266:550-52; Gefter et al. Somatic Cell Genet., cited supra;Lerner, Yale J. Biol. Med., cited supra; Kenneth, Monoclonal Antibodies,cited supra). Moreover, the ordinary skilled worker will appreciate thatthere are many variations of such methods which also would be useful.Typically, the immortal cell line (e.g., a myeloma cell line) is derivedfrom the same mammalian species as the lymphocytes. For example, murinehybridomas can be made by fusing lymphocytes from a mouse immunized withan immunogenic preparation of the present invention with an immortalizedmouse cell line. Preferred immortal cell lines are mouse myeloma celllines that are sensitive to culture medium containing hypoxanthine,aminopterin and thymidine (“HAT medium”). Any of a number of myelomacell lines may be used as a fusion partner according to standardtechniques, e.g., the P3-NS1/1-Ag4-1, P3-x63-Ag8.653 or Sp2/O-Ag14myeloma lines. These myeloma lines are available from the American TypeCulture Collection (ATCC), Rockville, Md. Typically, HAT-sensitive mousemyeloma cells are fused to mouse splenocytes using polyethylene glycol(“PEG”). Hybridoma cells resulting from the fusion are then selectedusing HAT medium, which kills unfused and unproductively fused myelomacells (unfused splenocytes die after several days because they are nottransformed). Hybridoma cells producing a monoclonal antibody thatspecifically binds the maf protein are identified by screening thehybridoma culture supernatants for such antibodies, e.g., using astandard ELISA assay.

Alternative to preparing monoclonal antibody-secreting hybridomas, amonoclonal anti-maf antibody can be identified and isolated by screeninga recombinant combinatorial immunoglobulin library (e.g., an antibodyphage display library) with a maf protein or peptide to thereby isolateimmunoglobulin library members that bind specifically to a Maf protein.Kits for generating and screening phage display libraries arecommercially available (e.g, the Pharmacia Recombinant Phage AntibodySystem, Catalog No. 27-9400-01; and the Stratagene SurfZAP™ PhageDisplay Kit, Catalog No. 240612). Additionally, examples of methods andreagents particularly amenable for use in generating and screeningantibody display library can be found in, for example, Ladner et al.U.S. Pat. No. 5,223,409; Kang et al. International Publication No. WO92/18619; Dower et al. International Publication No. WO 91/17271; Winteret al. International Publication WO 92/20791; Markland et al.International Publication No. WO 92/15679; Breitling et al.International Publication WO 93/01288; McCafferty et al. InternationalPublication No. WO 92/01047; Garrard et al. International PublicationNo. WO 92/09690; Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay etal. (1992) Hum Antibod Hybridomas 3:81-85; Huse et al. (1989) Science246:1275-1281; Griffiths et al. (1993) EMBO J 12:725-734; Hawkins et al.(1992) J Mol Biol 226:889-896; Clarkson et al. (1991) Nature352:624-628; Gram et al. (1992) PNAS 89:3576-3580; Garrad et al. (1991)Bio/Technology 9:1373-1377; Hoogenboom et al. (1991) Nuc Acid Res19:4133-4137; Barbas et al. (1991) PNAS 88:7978-7982; and McCafferty etal. Nature (1990) 348:552-554.

Once a monoclonal antibody specific for the Maf protein has beenidentified (e.g., either a hybridoma-derived monoclonal antibody or arecombinant antibody from a combinatorial library), DNAs encoding thelight and heavy chains of the monoclonal antibody are isolated bystandard molecular biology techniques. For hybridoma derived antibodies,light and heavy chain cDNAs can be obtained, for example, by PCRamplification or cDNA library screening. For recombinant antibodies,such as from a phage display library, cDNA encoding the light and heavychains can be recovered from the display package (e.g., phage) isolatedduring the library screening process. Nucleotide sequences of antibodylight and heavy chain genes from which PCR primers or cDNA libraryprobes can be prepared are known in the art. For example, many suchsequences are disclosed in Kabat, E. A., et al. (1991) Sequences ofProteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and Human Services, NIH Publication No. 91-3242 and in the“Vbase” human germline sequence database.

Once obtained, the antibody light and heavy chain sequences are clonedinto a recombinant expression vector using standard methods. Asdiscussed above, the sequences encoding the hydrophobic leaders of thelight and heavy chains are removed and sequences encoding a nuclearlocalization signal (e.g., from SV40 Large T antigen) are linkedin-frame to sequences encoding either the amino- or carboxy terminus ofboth the light and heavy chains. The expression vector can encode anintracellular antibody in one of several different forms. For example,in one embodiment, the vector encodes full-length antibody light andheavy chains such that a full-length antibody is expressedintracellularly. In another embodiment, the vector encodes a full-lengthlight chain but only the VH/CH1 region of the heavy chain such that aFab fragment is expressed intracellularly. In the most preferredembodiment, the vector encodes a single chain antibody (scFv) whereinthe variable regions of the light and heavy chains are linked by aflexible peptide linker (e.g., (Gly₄Ser)₃) and expressed as a singlechain molecule. To inhibit transcription factor activity in a cell, theexpression vector encoding the transcription factor-specificintracellular antibody is introduced into the cell by standardtransfection methods, as discussed hereinbefore.

Yet another form of an inhibitory agent of the invention is aninhibitory form of a Maf protein, also referred to herein as a dominantnegative inhibitor. The maf family of proteins are known to homodimerizeand to heterodimerize with other AP-1 family members, such as Fos andJun (see e.g., Kerppola, T. K. and Curran, T. (1994) Oncogene 9:675-684;Kataoka, K. et al. (1994) Mol. Cell. Biol. 14:700-712). One means toinhibit the activity of transcription factors that form dimers isthrough the use of a dominant negative inhibitor that has the ability todimerize with functional transcription factors but that lacks theability to activate transcription (see e.g., Petrak, D. et al. (1994) J.Immunol. 153:2046-2051). By dimerizing with functional transcriptionfactors, such dominant negative inhibitors can inhibit their functionalactivity. This process may occur naturally as a means to regulate geneexpression. For example, there are a number of “small” maf proteins,such as mafK, mafF, mafG and p18, which lack the amino terminal twothirds of c-Maf that contains the transactivating domain (Fujiwara, K.T. et al. (1993) Oncogene 8:2371-2380; Igarashi, K. et al. (1995) J.Biol. Chem. 270:7615-7624; Andrews, N. C. et al. (1993) Proc. Natl.Acad. Sci. USA 90:11488-11492; Kataoka, K. et al. (1995) Mol. Cell.Biol. 15:2180-2190). Homodimers of the small maf proteins act asnegative regulators of transcription (Igarashi, K. et al. (1994) Nature367:568-572) and three of the small maf proteins (MafK, MafF and MafG)have been shown to competitively inhibit transactivation mediated by thev-Maf oncoprotein (Kataoka, K. et al. (1996) Oncogene 12:53-62).Additionally, MafB has been identified as an interaction partner ofEts-1 and shown to inhibit Ets-1-mediated transactivation of thetransferrin receptor and to inhibit erythroid differentation (Sieweke,M. H. et al. (1996) Cell 85:49-60).

Accordingly, an inhibitory agent of the invention can be a form of a Mafprotein that has the ability to dimerize with c-Maf but that lacks theability to activate transcription. This dominant negative form of a Mafprotein may be, for example, a small Maf protein (e.g, MafK, MafF, MafG)that naturally lacks a transactivation domain, MafB or a mutated form ofc-Maf in which the transactivation domain has been removed. Suchdominant negative Maf proteins can be expressed in cells using arecombinant expression vector encoding the Maf protein, which isintroduced into the cell by standard transfection methods. To express amutant form of c-Maf lacking a transactivation domain, nucleotidesequences encoding the amino terminal transactivation domain of c-Mafare removed from the c-maf cDNA by standard recombinant DNA techniques.Preferably, at least amino acids 1-122 are removed. More preferably, atleast amino acids 1-187, or amino acids 1-257, are removed. Nucleotidesequences encoding the basic-leucine zipper region are maintained. Thetruncated cDNA is inserted into a recombinant expression vector, whichis then introduced into a cell to allow for expression of the truncatedc-maf, lacking a transactivation domain, in the cell.

Yet another type of inhibitory agent that can be used to inhibit theexpression and/or activity of a maf protein in a cell is chemicalcompound that inhibits the expression or activity of an endogenous maffamily protein in the cell. Such compounds can be identified usingscreening assays that select for compounds that inhibit the expressionor activity of a maf family protein. Examples of suitable screeningassays are described in further detail in subsection V below.

As discussed above with regard to stimulatory agents, the inhibitorymethods of the invention can involve the use of one or more additionalinhibitory agents that inhibit the expression or activity of one or moreadditional transcription factors that contributes to regulating theexpression of a Th1- or Th2-associated cytokine gene. For example, inone embodiment, the inhibitory method of the invention comprisescontacting a cell with a first agent that inhibits the expression oractivity a maf family protein and a second agent that inhibits theexpression or activity of an NF-AT protein. In another embodiment, theinhibitory method of the invention comprises contacting a cell with afirst agent that inhibits the expression or activity a maf familyprotein and a second agent that inhibits the expression or activity ofan AP-1 protein. In yet another embodiment, the inhibitory method of theinvention comprises contacting a cell with a first agent that inhibitsthe expression or activity a maf family protein, a second agent thatinhibits the expression or activity of an NF-AT protein and a thirdagent that inhibits the expression or activity of an AP-1 protein.Examples of types of inhibitory agents for inhibiting NF-AT or AP-1proteins include antisense nucleic acids, intracellular antibodies,dominant negative inhibitors and chemical compounds that inhibit theendogenous proteins, as described above. Regarding the latter, it isknown in the art that the nuclear translocation of NF-ATp is inhibitedby the immunosuppressive drugs cyclosporin A and FK506 (see e.g., Rao,A. (1994) Immunol. Today 15:274-281; Rao, A. (1995) J. Leukoc. Biol.57:536-542). Accordingly, in one embodiment of the inhibitory method, animmunosuppressive drug such as cyclosporin A or FK506 (or other relateddrug that inhibits the calcineurin pathway) is used in combination withan agent that inhibits the expression or activity of c-Maf.

The method of the invention for modulating production of Th2-associatedcytokines by a cell can be practiced either in vitro or in vivo (thelatter is discussed further in the following subsection). For practicingthe method in vitro, cells can be obtained from a subject by standardmethods and incubated (i.e., cultured) in vitro with a stimulatory orinhibitory agent of the invention to stimulate or inhibit, respectively,the production of Th2-associated cytokines. For example, peripheralblood mononuclear cells (PBMCs) can be obtained from a subject andisolated by density gradient centrifugation, e.g., with Ficoll/Hypaque.Specific cell populations can be depleted or enriched using standardmethods. For example, monocytes/macrophages can be isolated by adherenceon plastic. T cells or B cells can be enriched or depleted, for example,by positive and/or negative selection using antibodies to T cell or Bcell surface markers, for example by incubating cells with a specificprimary monoclonal antibody (mAb), followed by isolation of cells thatbind the mAb using magnetic beads coated with a secondary antibody thatbinds the primary mAb. Peripheral blood or bone marrow derivedhematopoietic stem cells can be isolated by similar techniques usingstem cell-specific mAbs (e.g., anti-CD34 mAbs). Specific cellpopulations can also be isolated by fluoresence activated cell sortingaccording to standard methods. Monoclonal antibodies to cell-specificsurface markers known in the art and many are commercially available.

When a stimulatory agent is used in vitro, resulting in stimulation ofthe production of Th2-associated cytokines, in particular IL-4, thecytokine(s) can be recovered from the culture supernatant for furtheruse. For example, the culture supernatant, or a purified fractionthereof, can be applied to T cells in culture to influence thedevelopment of Th1 or Th2 cells in vitro. Alternatively, the culturesupernatant, or a purified fraction thereof, can be administered to asubject to influence the development of Th1 vs. Th2 responses in thesubject.

Moreover, cells treated in vitro with either a stimulatory or inhibitoryagent can be administered to a subject to influence the development of aTh1 vs. Th2 response in the subject. Accordingly, in another embodiment,the method of the invention for modulating the production ofTh2-associated cytokines by a cell further comprises administering thecell to a subject to thereby modulate development of Th1 or Th2 cells ina subject. Preferred cell types for ex vivo modification andreadministration include T cells, B cells and hematopoietic stem cells.For administration to a subject, it may be preferable to first removeresidual agents in the culture from the cells before administering themto the subject. This can be done for example by a Ficoll/Hypaquegradient centrifugation of the cells. For further discussion of ex vivogenetic modification of cells followed by readministration to a subject,see also U.S. Pat. No. 5,399,346 by W. F. Anderson et al.

II. Methods for Modulating Development of Th1 or Th2 Cells in a Subject

Another aspect of the invention pertains to a method for modulatingdevelopment of Th1 or Th2 cells in a subject. The term “subject” isintended to include living organisms in which an immune response can beelicited. Preferred subjects are mammals. Examples of subjects includehumans, monkeys, dogs, cats, mice, rats, cows, horses, goats and sheep.As discussed above, one way to modulate Th1/Th2 ratios in a subject isto treat cells (e.g., T cells, B cells or hematopoietic stem cells) exvivo with one or more modulatory agents of the invention, such thatproduction of a Th2-associated cytokine by the cells is modulated,followed by administration of the cells to the subject. In anotherembodiment, Th1/Th2 ratios are modulated in a subject by administeringto the subject an agent that modulates the activity of a transcriptionfactor that regulates expression of a Th2-associated cytokine gene suchthat development of Th1 or Th2 cells in the subject is modulated.Preferably, the transcription factor is a maf family protein and mostpreferably a c-Maf protein. Preferably, the Th2-associated cytokine isIL-4. Development of a Th2 response in the subject can be promoted byadministration of one or more stimulatory agents of the invention,whereas development of a Th1 response in the subject can be promoted byadministration of one or more inhibitory agents of the invention. Asdiscussed above, in certain situations it may be desirable, in additionto modulating the activity of a maf family protein, to also modulate theactivity of other transcription factors involved in regulating Th1- orTh2-associated cytokine genes. Preferably, the activity of an NF-ATprotein(s) is also modulated. Additionally or alternatively, theactivity of an AP-1 protein(s) is also modulated.

For stimulatory or inhibitory agents that comprise nucleic acids(including recombinant expression vectors encoding transcriptionfactors, antisense RNA, intracellular antibodies or dominant negativeinhibitors), the agents can be introduced into cells of the subjectusing methods known in the art for introducing nucleic acid (e.g., DNA)into cells in vivo. Examples of such methods include:

Direct Injection: Naked DNA can be introduced into cells in vivo bydirectly injecting the DNA into the cells (see e.g., Acsadi et al.(1991) Nature 332:815-818; Wolff et al. (1990) Science 247:1465-1468).For example, a delivery apparatus (e.g., a “gene gun”) for injecting DNAinto cells in vivo can be used. Such an apparatus is commerciallyavailable (e.g., from BioRad).

Receptor-Mediated DNA Uptake: Naked DNA can also be introduced intocells in vivo by complexing the DNA to a cation, such as polylysine,which is coupled to a ligand for a cell-surface receptor (see forexample Wu, G. and Wu, C. H. (1988) J. Biol. Chem. 263:14621; Wilson etal. (1992) J. Biol. Chem. 267:963-967; and U.S. Pat. No. 5,166,320).Binding of the DNA-ligand complex to the receptor facilitates uptake ofthe DNA by receptor-mediated endocytosis. A DNA-ligand complex linked toadenovirus capsids which naturally disrupt endosomes, thereby releasingmaterial into the cytoplasm can be used to avoid degradation of thecomplex by intracellular lysosomes (see for example Curiel et al. (1991)Proc. Natl. Acad. Sci. USA 88:8850; Cristiano et al. (1993) Proc. Natl.Acad Sci. USA 90:2122-2126).

Retroviruses: Defective retroviruses are well characterized for use ingene transfer for gene therapy purposes (for a review see Miller, A. D.(1990) Blood 76:271). A recombinant retrovirus can be constructed havinga nucleotide sequences of interest incorporated into the retroviralgenome. Additionally, portions of the retroviral genome can be removedto render the retrovirus replication defective. The replicationdefective retrovirus is then packaged into virions which can be used toinfect a target cell through the use of a helper virus by standardtechniques. Protocols for producing recombinant retroviruses and forinfecting cells in vitro or in vivo with such viruses can be found inCurrent Protocols in Molecular Biology, Ausubel, F. M. et al. (eds.)Greene Publishing Associates, (1989), Sections 9.10-9.14 and otherstandard laboratory manuals. Examples of suitable retroviruses includepLJ, pZIP, pWE and pEM which are well known to those skilled in the art.Examples of suitable packaging virus lines include ψCrip, ψCre, ψ2 andψAm. Retroviruses have been used to introduce a variety of genes intomany different cell types, including epithelial cells, endothelialcells, lymphocytes, myoblasts, hepatocytes, bone marrow cells, in vitroand/or in vivo (see for example Eglitis, et al. (1985) Science230:1395-1398; Danos and Mulligan (1988) Proc. Natl. Acad. Sci. USA85:6460-6464; Wilson et al. (1988) Proc. Natl. Acad. Sci. USA85:3014-3018; Armentano et al. (1990) Proc. Natl. Acad. Sci. USA87:6141-6145; Huber et al. (1991) Proc. Natl. Acad. Sci. USA88:8039-8043; Ferry et al. (1991) Proc. Natl. Acad. Sci. USA88:8377-8381; Chowdhury et al. (1991) Science 254:1802-1805; vanBeusechem et al. (1992) Proc. Natl. Acad. Sci. USA 89:7640-7644; Kay etal. (1992) Human Gene Therapy 3:641-647; Dai et al. (1992) Proc. Natl.Acad. Sci. USA 89:10892-10895; Hwu et al. (1993) J. Immunol.150:4104-4115; U.S. Pat. No. 4,868,116; U.S. Pat. No. 4,980,286; PCTApplication WO 89/07136; PCT Application WO 89/02468; PCT Application WO89/05345; and PCT Application WO 92/07573). Retroviral vectors requiretarget cell division in order for the retroviral genome (and foreignnucleic acid inserted into it) to be integrated into the host genome tostably introduce nucleic acid into the cell. Thus, it may be necessaryto stimulate replication of the target cell.

Adenoviruses: The genome of art adenovirus can be manipulated such thatit encodes and expresses a gene product of interest but is inactivatedin terms of its ability to replicate in a normal lytic viral life cycle.See for example Berkner et al. (1988) BioTechniques 6:616; Rosenfeld etal. (1991) Science 252:431-434; and Rosenfeld et al. (1992) Cell68:143-155. Suitable adenoviral vectors derived from the adenovirusstrain Ad type 5 dl324 or other strains of adenovirus (e.g., Ad2, Ad3,Ad7 etc.) are well known to those skilled in the art. Recombinantadenoviruses are advantageous in that they do not require dividing cellsto be effective gene delivery vehicles and can be used to infect a widevariety of cell types, including airway epithelium (Rosenfeld et al.(1992) cited supra), endothelial cells (Lemarchand et al. (1992) Proc.Natl. Acad. Sci. USA 89:6482-6486), hepatocytes (Herz and Gerard (1993)Proc. Natl. Acad. Sci. USA 90:2812-2816) and muscle cells (Quantin etal. (1992) Proc. Natl. Acad. Sci. USA 89:2581-2584). Additionally,introduced adenoviral DNA (and foreign DNA contained therein) is notintegrated into the genome of a host cell but remains episomal, therebyavoiding potential problems that can occur as a result of insertionalmutagenesis in situations where introduced DNA becomes integrated intothe host genome (e.g., retroviral DNA). Moreover, the carrying capacityof the adenoviral genome for foreign DNA is large (up to 8 kilobases)relative to other gene delivery vectors (Berkner et al. cited supra;Haj-Ahmand and Graham (1986) J. Virol. 57:267). Mostreplication-defective adenoviral vectors currently in use are deletedfor all or parts of the viral E1 and E3 genes but retain as much as 80%of the adenoviral genetic material.

Adeno-Associated Viruses: Adeno-associated virus (AAV) is a naturallyoccurring defective virus that requires another virus, such as anadenovirus or a herpes virus, as a helper virus for efficientreplication and a productive life cycle. (For a review see Muzyczka etal. Curr. Topics in Micro. and Immunol. (1992) 158:97-129). It is alsoone of the few viruses that may integrate its DNA into non-dividingcells, and exhibits a high frequency of stable integration (see forexample Flotte et al. (1992) Am. J. Respir. Cell. Mol. Biol. 7:349-356;Samulski et al. (1989) J. Virol. 63:3822-3828; and McLaughlin et al.(1989) J. Virol. 62:1963-1973). Vectors containing as little as 300 basepairs of AAV can be packaged and can integrate. Space for exogenous DNAis limited to about 4.5 kb. An AAV vector such as that described inTratschin et al. (1985) Mol. Cell. Biol. 5:3251-3260 can be used tointroduce DNA into cells. A variety of nucleic acids have beenintroduced into different cell types using AAV vectors (see for exampleHermonat et al. (1984) Proc. Natl. Acad. Sci. USA 81:6466-6470;Tratschin et al. (1985) Mol. Cell. Biol. 4:2072-2081; Wondisford et al.(1988) Mol. Endocrinol. 2:32-39; Tratschin et al. (1984) J. Virol.51:611-619; and Flotte et al. (1993) J. Biol. Chem. 268:3781-3790).

The efficacy of a particular expression vector system and method ofintroducing nucleic acid into a cell can be assessed by standardapproaches routinely used in the art. For example, DNA introduced into acell can be detected by a filter hybridization technique (e.g., Southernblotting) and RNA produced by transcription of introduced DNA can bedetected, for example, by Northern blotting, RNase protection or reversetranscriptase-polymerase chain reaction (RT-PCR). The gene product canbe detected by an appropriate assay, for example by immunologicaldetection of a produced protein, such as with a specific antibody, or bya functional assay to detect a functional activity of the gene product,such as an enzymatic assay.

A modulatory agent, such as a chemical compound that stimulates orinhibits endogenous c-Maf activity, can be administered to a subject asa pharmaceutical composition. Such compositions typically comprise themodulatory agent and a pharmaceutically acceptable carrier. As usedherein the term “pharmaceutically acceptable carrier” is intended toinclude any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents, and thelike, compatible with pharmaceutical administration. The use of suchmedia and agents for pharmaceutically active substances is well known inthe art. Except insofar as any conventional media or agent isincompatible with the active compound, use thereof in the compositionsis contemplated. Supplementary active compounds can also be incorporatedinto the compositions.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. For example,solutions or suspensions used for parenteral, intradermal, orsubcutaneous application can include the following components: a sterilediluent such as water for injection, saline solution, fixed oils,polyethylene glycols, glycerine, propylene glycol or other syntheticsolvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates or phosphates and agents for the adjustment oftonicity such as sodium chloride or dextrose. pH can be adjusted withacids or bases, such as hydrochloric acid or sodium hydroxide. Theparenteral preparation can be enclosed in ampoules, disposable syringesor multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyetheylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying which yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These may be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on (a) the unique characteristics of the active compound andthe particular therapeutic effect to be achieved, and (b) thelimitations inherent in the art of compounding such an active compoundfor the treatment of individuals.

III. Applications of the Methods of the Invention

Identification of the transcription factor that controls the productionof IL-4, and hence continued formation of Th2 cells, allows forselective manipulation of T cell subsets in a variety of clinicalsituations using the modulatory methods of the invention. Thestimulatory methods of the invention (i.e., methods that use astimulatory agent) result in production of Th2-associated cytokines,with concomitant promotion of a Th2 response and downregulation of a Th1response. In contrast, the inhibitory methods of the invention (i.e.,methods that use an inhibitory agent) inhibit the production ofTh2-associated cytokines, with concomitant downregulation of a Th2response and promotion of a Th1 response. Thus, to treat a diseasecondition wherein a Th2 response is beneficial, a stimulatory method ofthe invention is selected such that Th2 responses are promoted whiledownregulating Th1 responses. Alternatively, to treat a diseasecondition wherein a Th1 response is beneficial, an inhibitory method ofthe invention is selected such that Th2 responses are downregulatedwhile promoting Th1 responses. Application of the methods of theinvention to the treatment of disease conditions may result in cure ofthe condition, a decrease in the type or number of symptoms associatedwith the condition, either in the long term or short term (i.e.,amelioration of the condition) or simply a transient beneficial effectto the subject.

Numerous disease conditions associated with a predominant Th1 orTh2-type response have been identified and could benefit from modulationof the type of response mounted in the individual suffering from thedisease condition. Application of the immunomodulatory methods of theinvention to such diseases is described in further detail below.

A. Allergies

Allergies are mediated through IgE antibodies whose production isregulated by the activity of Th2 cells and the cytokines producedthereby. In allergic reactions, IL-4 is produced by Th2 cells, whichfurther stimulates production of IgE antibodies and activation of cellsthat mediate allergic reactions, i.e., mast cells and basophils. IL-4also plays an important role in eosinophil mediated inflammatoryreactions. Accordingly, the inhibitory methods of the invention can beused to inhibit the production of Th2-associated cytokines, and inparticular IL-4, in allergic patients as a means to downregulateproduction of pathogenic IgE antibodies. An inhibitory agent may bedirectly administered to the subject or cells (e.g., Thp cells or Th2cells) may be obtained from the subject, contacted with an inhibitoryagent ex vivo, and readministered to the subject. Moreover, in certainsituations it may be beneficial to coadminister to the subject theallergen together with the inhibitory agent or cells treated with theinhibitory agent to inhibit (e.g., desensitize) the allergen-specificresponse. The treatment may be further enhanced by administering otherTh1-promoting agents, such as the cytokine IL-12 or antibodies toTh2-associated cytokines (e.g., anti-IL-4 antibodies), to the allergicsubject in amounts sufficient to further stimulate a Th1-type response.

B. Cancer

The expression of Th2-promoting cytokines has been reported to beelevated in cancer patients (see e.g., Yamamura, M., et al. (1993) J.Clin. Invest. 91:1005-1010; Pisa, P., et al. (1992) Proc. Natl. Acad.Sci. USA 89:7708-7712) and malignant disease is often associated with ashift from Th1 type responses to Th2 type responses along with aworsening of the course of the disease. Accordingly, the inhibitorymethods of the invention can be used to inhibit the production ofTh2-associated cytokines in cancer patients, as a means to counteractthe Th1 to Th2 shift and thereby promote an ongoing Th1 response in thepatients to ameliorate the course of the disease. The inhibitory methodcan involve either direct administration of an inhibitory agent to asubject with cancer or ex vivo treatment of cells obtained from thesubject (e.g., Thp or Th2 cells) with an inhibitory agent followed byreadministration of the cells to the subject. The treatment may befurther enhanced by administering other Th1-promoting agents, such asthe cytokine IL-12 or antibodies to Th2-associated cytokines (e.g.,anti-IL-4 antibodies), to the recipient in amounts sufficient to furtherstimulate a Th1-type response.

C. Infectious Diseases

The expression of Th2-promoting cytokines also has been reported toincrease during a variety of infectious diseases, including HIVinfection, tuberculosis, leishmaniasis, schistosomiasis, filarialnematode infection and intestinal nematode infection (see e.g.; Shearer,G. M. and Clerici, M. (1992) Prog. Chem. Immunol. 54:21-43; Clerici, Mand Shearer, G. M. (1993) Immunology Today 14:107-111; Fausi, A. S.(1988) Science 239:617-623; Locksley, R. M. and Scott, P. (1992)Immunoparasitology Today 1:A58-A61; Pearce, E. J., et al. (1991) J. Exp.Med. 173:159-166; Grzych, J-M., et al. (1991) J. Immunol. 141:1322-1327;Kullberg, M. C., et al. (1992) J. Immunol. 148:3264-3270; Bancroft, A.J., et al. (1993) J. Immunol. 150:1395-1402; Pearlman, E., et al. (1993)Infect. Immun. 61:1105-1112; Else, K. J., et al. (1994) J. Exp. Med.179:347-351) and such infectious diseases are also associated with a Th1to Th2 shift in the immune response. Accordingly, the inhibitory methodsof the invention can be used to inhibit the production of Th2-associatedcytokines in subjects with infectious diseases, as a means to counteractthe Th1 to Th2 shift and thereby promote an ongoing Th1 response in thepatients to ameliorate the course of the infection. The inhibitorymethod can involve either direct administration of an inhibitory agentto a subject with an infectious disease or ex vivo treatment of cellsobtained from the subject (e.g., Thp or Th2 cells) with an inhibitoryagent followed by readministration of the cells to the subject. Thetreatment may be further enhanced by administering other Th1-promotingagents, such as the cytokine IL-12 or antibodies to Th2-associatedcytokines (e.g., anti-IL-4 antibodies), to the recipient in amountssufficient to further stimulate a Th1-type response.

D. Autoimmune Diseases

The stimulatory methods of the invention can be used therapeutically inthe treatment of autoimmune diseases that are associated with a Th2-typedysfunction. Many autoimmune disorders are the result of inappropriateactivation of T cells that are reactive against self tissue and thatpromote the production of cytokines and autoantibodies involved in thepathology of the diseases. Modulation of T helper-type responses canhave an effect on the course of the autoimmune disease. For example, inexperimental allergic encephalomyelitis (EAE), stimulation of a Th2-typeresponse by administration of IL-4 at the time of the induction of thedisease diminishes the intensity of the autoimmune disease (Paul, W. E.,et al. (1994) Cell 76:241-251). Furthermore, recovery of the animalsfrom the disease has been shown to be associated with an increase in aTh2-type response as evidenced by an increase of Th2-specific cytokines(Koury, S. J., et al. (1992) J. Exp. Med. 176:1355-1364). Moreover, Tcells that can suppress EAE secrete Th2-specific cytokines (Chen, C., etal. (1994) Immunity 1:147-154). Since stimulation of a Th2-type responsein EAE has a protective effect against the disease, stimulation of a Th2response in subjects with multiple sclerosis (for which EAE is a model)is likely to be beneficial therapeutically.

Similarly, stimulation of a Th2-type response in type I diabetes in miceprovides a protective effect against the disease. Indeed, treatment ofNOD mice with IL-4 (which promotes a Th2 response) prevents or delaysonset of type I diabetes that normally develops in these mice (Rapoport,M. J., et al. (1993) J. Exp. Med. 178:87-99). Thus, stimulation of a Th2response in a subject suffering from or susceptible to diabetes mayameliorate the effects of the disease or inhibit the onset of thedisease.

Yet another autoimmune disease in which stimulation of a Th2-typeresponse may be beneficial is rheumatoid arthritis (RA). Studies haveshown that patients with rheumatoid arthritis have predominantly Th1cells in synovial tissue (Simon, A. K., et al., (1994) Proc. Natl. Acad.Sci. USA 91:8562-8566). By stimulating a Th2 response in a subject withRA, the detrimental Th1 response can be concomitantly downmodulated tothereby ameliorate the effects of the disease.

Accordingly, the stimulatory methods of the invention can be used tostimulate production of Th2-associated cytokines in subjects sufferingfrom, or susceptible to, an autoimmune disease in which a Th2-typeresponse is beneficial to the course of the disease. The stimulatorymethod can involve either direct administration of a stimulatory agentto the subject or ex vivo treatment of cells obtained from the subject(e.g., Thp, Th1 cells, B cells, non-lymphoid cells) with a stimulatoryagent followed by readministration of the cells to the subject. Thetreatment may be further enhanced by administering other Th2-promotingagents, such as IL-4 itself or antibodies to Th1-associated cytokines,to the subject in amounts sufficient to further stimulate a Th2-typeresponse.

In contrast to the autoimmune diseases described above in which a Th2response is desirable, other autoimmune diseases may be ameliorated by aTh1-type response. Such diseases can be treated using an inhibitoryagent of the invention (as described above for cancer and infectiousdiseases). The treatment may be further enhanced by administrating aTh1-promoting cytokine (e.g., IFN-γ) to the subject in amountssufficient to further stimulate a Th1-type response.

The efficacy of agents for treating autoimmune diseases can be tested inthe above described animal models of human diseases (e.g., EAE as amodel of multiple sclerosis and the NOD mice as a model for diabetes) orother well characterized animal models of human autoimmune diseases.Such animal models include the mrl/lpr/lpr mouse as a model for lupuserythematosus, murine collagen-induced arthritis as a model forrheumatoid arthritis, and murine experimental myasthenia gravis (seePaul ed., Fundamental Immunology, Raven Press, New York, 1989, pp.840-856). A modulatory (i.e., stimulatory or inhibitory) agent of theinvention is administered to test animals and the course of the diseasein the test animals is then monitored by the standard methods for theparticular model being used. Effectiveness of the modulatory agent isevidenced by amelioration of the disease condition in animals treatedwith the agent as compared to untreated animals (or animals treated witha control agent).

Non-limiting examples of autoimmune diseases and disorders having anautoimmune component that may be treated according to the inventioninclude diabetes mellitus, arthritis (including rheumatoid arthritis,juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis),multiple sclerosis, myasthenia gravis, systemic lupus erythematosis,autoimmune thyroiditis, dermatitis (including atopic dermatitis andeczematous dermatitis), psoriasis, Sjögren's Syndrome, includingkeratoconjunctivitis sicca secondary to Sjögren's Syndrome, alopeciaareata, allergic responses due to arthropod bite reactions, Crohn'sdisease, aphthous ulcer, iritis, conjunctivitis, keratoconjunctivitis,ulcerative colitis, asthma, allergic asthma, cutaneous lupuserythematosus, scleroderma, vaginitis, proctitis, drug eruptions,leprosy reversal reactions, erythema nodosum leprosum, autoimmuneuveitis, allergic encephalomyelitis, acute necrotizing hemorrhagicencephalopathy, idiopathic bilateral progressive sensorineural hearingloss, aplastic anemia, pure red cell anemia, idiopathicthrombocytopenia, polychondritis, Wegener's granulomatosis, chronicactive hepatitis, Stevens-Johnson syndrome, idiopathic sprue, lichenplanus, Crohn's disease, Graves ophthalmopathy, sarcoidosis, primarybiliary cirrhosis, uveitis posterior, and interstitial lung fibrosis.

E. Transplantation

While graft rejection or graft acceptance may not be attributableexclusively to the action of a particular T cell subset (i.e., Th1 orTh2 cells) in the graft recipient (for a discussion see Dallman, M. J.(1995) Curr. Opin. Immunol. 7:632-638), numerous studies have implicateda predominant Th2 response in prolonged graft survival or a predominantTh2 response in graft rejection. For example, graft acceptance has beenassociated with production of a Th2 cytokine pattern and/or graftrejection has been associated with production of a Th1 cytokine pattern(see e.g., Takeuchi, T. et al. (1992) Transplantation 53:1281-1291;Tzakis, A. G. et al. (1994) J. Pediatr. Surg. 29:754-756; Thai, N. L. etal. (1995) Transplantation 59:274-281). Additionally, adoptive transferof cells having a Th2 cytokine phenotype prolongs skin graft survival(Maeda, H. et al. (1994) Int. Immunol. 6:855-862) and reducesgraft-versus-host disease (Fowler, D. H. et al. (1994) Blood84:3540-3549; Fowler, D. H. et al. (1994) Prog. Clin. Biol. Res.389:533-540). Still further, administration of IL-4, which promotes Th2differentiation, prolongs cardiac allograft survival (Levy, A. E. andAlexander, J. W. (1995) Transplantation 60:405-406), whereasadministration of IL-12 in combination with anti-IL-10 antibodies, whichpromotes Th1 differentiation, enhances skin allograft rejection(Gorczynski, R. M. et al. (1995) Transplantation 60:1337-1341).

Accordingly, the stimulatory methods of the invention can be used tostimulate production of Th2-associated cytokines in transplantrecipients to prolong survival of the graft. The stimulatory methods canbe used both in solid organ transplantation and in bone marrowtransplantation (e.g., to inhibit graft-versus-host disease). Thestimulatory method can involve either direct administration of astimulatory agent to the transplant recipient or ex vivo treatment ofcells obtained from the subject (e.g., Thp, Th1 cells, B cells,non-lymphoid cells) with a stimulatory agent followed byreadministration of the cells to the subject. The treatment may befurther enhanced by administering other Th2-promoting agents, such asIL-4 itself or antibodies to Th1-associated cytokines, to the recipientin amounts sufficient to further stimulate a Th2-type response.

In addition to the foregoing disease situations, the modulatory methodsof the invention also are useful for other purposes. For example, thestimulatory methods of the invention (i.e., methods using a stimulatoryagent) can be used to stimulate production of Th2-promoting cytokines(e.g., IL-4) in vitro for commercial production of these cytokines(e.g., cells can be contacted with the stimulatory agent in vitro tostimulate IL-4 production and the IL-4 can be recovered from the culturesupernatant, further purified if necessary, and packaged for commercialuse).

Furthermore, the modulatory methods of the invention can be applied tovaccinations to promote either a Th1 or a Th2 response to an antigen ofinterest in a subject. That is, the agents of the invention can serve asadjuvants to direct an immune response to a vaccine either to a Th1response or a Th2 response. For example, to stimulate an antibodyresponse to an antigen of interest (i.e., for vaccination purposes), theantigen and a stimulatory agent of the invention can be coadministeredto a subject to promote a Th2 response to the antigen in the subject,since Th2 responses provide efficient B cell help and promote IgG1production. Alternatively, to promote a cellular immune response to anantigen of interest, the antigen and an inhibitory agent of theinvention can be coadministered to a subject to promote a Th1 responseto the antigen in a subject, since Th1 responses favor the developmentof cell-mediated immune responses (e.g., delayed hypersensitivityresponses). The antigen of interest and the modulatory agent can beformulated together into a single pharmaceutical composition or inseparate compositions. In a preferred embodiment, the antigen ofinterest and the modulatory agent are administered simultaneously to thesubject. Alternatively, in certain situations it may be desirable toadminister the antigen first and then the modulatory agent or vice versa(for example, in the case of an antigen that naturally evokes a Th1response, it may be beneficial to first administer the antigen alone tostimulate a Th1 response and then administer a stimulatory agent, aloneor together with a boost of antigen, to shift the immune response to aTh2 response).

IV. Compositions for Modulating Th2-Associated Cytokine Production

Another aspect of the invention pertains to compositions that can beused to modulate Th2-associated cytokine production by a cell or Th1/Th2development in a subject in accordance with the methods of theinvention. The invention provides recombinant expression vectorscomprising a nucleotide sequence encoding a maf family proteinoperatively linked to regulatory sequences that direct expression of themaf family protein specifically in certain cell types. In a preferredembodiment, the regulatory sequences direct expression of the maf familyprotein specifically in lymphoid cells (e.g., T cells or B cells). Inone embodiment, the lymphoid cells are T cells. T cell specificregulatory elements are known in the art, such as the promoterregulatory region of T cell receptor genes (see e.g., Winoto andBaltimore (1989) EMBO J. 8:729-733; Leiden, J. M. (1994) Annu. Rev.Immunol. 11:539-570; Hettman, T. and Cohen, A. (1994) Mol. Immunol.31:315-322; Redondo, J. M. et al. (1991) Mol. Cell. Biol. 11:5671-5680).Other examples of T cell specific regulatory elements are those derivedfrom the CD3 gene (see e.g., Clevers, H. et al. (1988) Proc. Natl. Acad.Sci. USA 85:8623-8627; Clevers, H. C. et al. (1988) Proc. Natl. Acad.Sci. USA 85:8156-8160; Georgopoulos, K. et al. (1988) EMBO J.7:2401-2407), the CD4 gene (see e.g., Sawada, S. and Littman, D. R.(1991) Mol. Cell. Biol. 11:5506-5515; Salmon, P. et al. (1993) Proc.Natl. Acad. Sci. USA 90:7739-7743; Hanna, Z. et al. (1994) Mol. Cell.Biol. 14:1084-1094; see also GenBank accession numbers U01066 and S68043for human CD4 promoter sequences) and the CD2 gene (see e.g.,Zhumabekov, T. et al. (1995) J. Immunol. Methods 185:133-140). A DNAfragment comprising one or more T cell specific regulatory elements,such as a promoter and enhancer of a T cell receptor gene, can beobtained by standard molecular biology methods, such as by PCR usingoligonucleotide primers corresponding to the 5′ and 3′ ends of thedesired region and genomic DNA from T cells as the template. Once theDNA fragment comprising T cell specific regulatory elements is obtained,it can be operatively linked to a cDNA encoding a maf protein (e.g., thetwo DNA fragments can be ligated together such that the regulatoryelements are located 5′ of the maf sequences) and introduced intovector, such as a plasmid vector, using standard molecular biologytechniques.

In another embodiment, the lymphoid cells are B cells (i.e., within therecombinant expression vector the nucleotide sequences encoding a maffamily protein are operatively linked to regulatory sequences thatdirect expression of the maf family specifically in B cells). B cellspecific regulatory elements are known in the art, such as the promoterregulatory region of immunoglobulin genes (see e.g., Banerji et al.(1983) Cell 33:729-740; Queen and Baltimore (1983) Cell 33:741-748).Other examples of B cell specific regulatory elements are those derivedfrom the CD20 (B1) gene (see e.g., Thevenin, C. et al. (1993) J. Biol.Chem. 268:5949-5956; Rieckmann, P. et al. (1991) J. Immunol.147:3994-3999), the Fc epsilon RIIa gene (see e.g., Suter, U. et al.(1989) J. Immunol. 143:3087-3092) and major histocompatibility class IIgenes (see e.g, Glimcher, L. H. and Kara, C. J. (1992) Annu. Rev.Immunol. 10:13-49; Benoist, C. and Mathis, D. (1990) Annu. Rev. Immunol.8:681-715). A DNA fragment comprising B cell specific regulatoryelements, such as a promoter and enhancer of an immunoglobulin gene, canbe obtained by standard molecular biology methods, such as by PCR usingoligonucleotide primers corresponding to the 5′ and 3′ ends of thedesired region and genomic DNA from B cells as the template. Once theDNA fragment comprising B cell specific regulatory elements is obtained,it can be operatively linked to a cDNA encoding a maf protein (e.g., thetwo DNA fragments can be ligated together such that the regulatoryelements are located 5′ of the maf sequences) and introduced intovector, such as a plasmid vector, using standard molecular biologytechniques.

In yet another embodiment, the invention provides recombinant expressionvectors comprising a nucleotide sequence encoding a maf family proteinoperatively linked to regulatory sequences that direct expression of themaf family protein specifically in hematopoietic stem cells.Hematopoietic stem cell specific regulatory elements are known in theart. Preferably regulatory elements derived from the CD34 gene are used(see e.g., Satterthwaite, A. B. et al. (1992) Genomics 12:788-794; Burn,T. C. et al. (1992) Blood 80:3051-3059).

Another aspect of the invention pertains to recombinant host cells thatexpress a maf family protein. Such host cells can be used to produce aTh2-associated cytokine (e.g., IL-4). Such host cells also can beadministered to a subject to produce a Th2-associated cytokine in thesubject as a means to manipulate Th1:Th2 ratios in the subject. Theterms “host cell” and “recombinant host cell” are used interchangeablyherein to refer to a cell into which a recombinant expression vector hasbeen introduced. It is understood that such terms refer not only to theparticular subject cell but to the progeny of such a cell. Becausecertain modifications may occur in succeeding generations due to eithermutation or environmental influences, such progeny may not, in fact, beidentical to the parent cell, but as long as these progeny cells retainthe recombinant expression vector, these progeny are still intended tobe included within the scope of the term “host cell” as used herein.

In one embodiment, the invention provides a host lymphoic cell intowhich a recombinant expression vector encoding a maf family protein hasbeen introduced. The host lymphoid cell can be a T cell or a B cell. Ahost T cell of the invention can be, for example a T cell clone that iscultured in vitro (such as those described in the Examples) or,alternatively, a normal T cell that is isolated from a subject (e.g., aperipheral blood T cell or a splenic T cell). Standard methods forpreparing and culturing T cell clones in vitro, or isolating T cells(e.g., from peripheral blood) are known in the art, for example throughthe use of mAbs that bind T cell specific cell surface markers (e.g.,CD3) or surface markers for specific subsets of T cells (e.g., CD4 orCD8). The recombinant expression vector can be introduced into the Tcell by one of a variety of known transfection methods for introducingDNA into mammalian cells, including calcium phosphate or calciumchloride co-precipitation, DEAE-dextran-mediated transfection,lipofection, or electroporation. Suitable methods for transforming ortransfecting host cells can be found in Sambrook et al. (MolecularCloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratorypress (1989)), and other laboratory manuals.

In another embodiment, the host lymphoid cell of the invention is a hostB cell into which a recombinant expression vector encoding a maf familyprotein has been introduced. The B cell can be, for example a B lymphomacell that is cultured in vitro (such as M12 cells as described in theExamples) or, alternatively, a normal B cell that is isolated from asubject (e.g., a peripheral blood B cell or a splenic B cell). Various Blymphoma cell lines are available in the art and standard methods forculturing such cells in vitro are known. Additionally, standard methodsfor isolating normal B cells (e.g., from peripheral blood) are known inthe art, for example through the use of mAbs that bind B cell specificcell surface markers (e.g., membrane immunoglobulin, B7-1, CD20). Therecombinant expression vector can be introduced into the B cell bystandard methods, as described above for T cells.

In yet another embodiment, the invention provides a host hematopoieticstem cell into which a recombinant expression vector encoding a maffamily protein has been introduced. Hematopoietic stem cells can beisolated from a subject (e.g., from peripheral blood or bone marrow ofthe subject) using standard methods known in the art for isolating suchstem cells, for example through the use of mAbs that bind hematopoieticstem cell specific cell surface markers, preferably CD34 (for furtherdescriptions of isolation of stem cells, see e.g., Wagner, J. E. et al.(1995) Blood 86:512-523; Murray, L. et al. (1995) Blood 85:368-378;Bernardi, A. C. et al. (1995) Science 267:104-108; Bernstein, I. D. etal. (1994) Blood Cells 20:15-24; Angelini, A. et al. (1993) Int. J.Artif. Organs 16 Suppl. 5:13-18; Kato, K. and Radburch, A. (1993)Cytometry 14:384-392; Lebkowski, J. S. et al. (1992) Transplantation53:1011-1019; Lebkowski, J. et al. (1993) J. Hematother. 2:339-342). Therecombinant expression vector can be introduced into the hematopoieticstem cell by standard methods, as described above for T cells.

V. Screening Assays

Another aspect of the invention pertains to screening assays foridentifying compounds that modulate the activity of a transcriptionfactor that regulates expression of a Th2-associated cytokine gene. Invarious embodiments, these screening assays can identify, for example,compounds that modulate the expression or functional activity of thetranscription factor, proteins that interact with the transcriptionfactor, as well as compounds that modulate these protein-proteininteractions, and compounds that modulate the interaction of thetranscription factor with a MARE within a Th2-associated cytokine gene.

In a preferred embodiment, the invention provides a method comprising:

a) preparing an indicator cell, wherein said indicator cell contains:

-   -   i) a recombinant expression vector encoding a transcription        factor that regulates expression of a Th2-associated cytokine        gene; and    -   ii) a vector comprising regulatory sequences of the        Th2-associated cytokine gene operatively linked a reporter gene;

b) contacting the indicator cell with a test compound;

c) determining the level of expression of the reporter gene in theindicator cell in the presence of the test compound;

d) comparing the level of expression of the reporter gene in theindicator cell in the presence of the test compound with the level ofexpression of the reporter gene in the indicator cell in the absence ofthe test compound; and

e) identifying a compound that modulates the activity of a transcriptionfactor that regulates expression of a Th2-associated cytokine gene.

Preferably, the transcription factor is a member of the maf family andmost preferably a c-Maf protein. Recombinant expression vectors that canbe used for expression of a c-Maf protein in an indicator cell are knownin the art (see discussions above and also the Examples). In oneembodiment, within the expression vector the c-Maf coding sequences areoperatively linked to regulatory sequences that allow for constitutiveexpression of c-Maf in the indicator cell (e.g., viral regulatorysequences, such as a cytomegalovirus promoter/enhancer, can be used).Use of a recombinant expression vector that allows for constitutiveexpression of c-Maf in the indicator cell is preferred foridentification of compounds that enhance or inhibit the activity ofc-Maf. In an alternative embodiment, within the expression vector thec-Maf coding sequences are operatively linked to regulatory sequences ofthe c-maf gene (i.e., the promoter regulatory region derived from theendogenous c-maf gene). Use of a recombinant expression vector in whichc-Maf protein expression is controlled by c-maf regulatory sequences ispreferred for identification of compounds that enhance or inhibit thetranscriptional expression of c-Maf.

Preferably, the Th2-associated cytokine is interleukin-4. It haspreviously shown that Th2-specific, inducible IL-4 expression can bedirected by as little as 157 bp of the proximal IL-4 promoter in Th2cells (Hodge, M. et al. (1995) J. Immunol. 154:6397-6405). Accordingly,in one embodiment, the method utilizes a reporter gene constructcontaining this region of the proximal IL-4 promoter, most preferablynucleotides −157 to +58 (relative to the start site of transcription at+1) of the IL-4 promoter. Alternatively, stronger reporter geneexpression can be achieved using a longer portion of the IL-4 upstreamregulatory region, such as about 3 kb of upstream regulatory sequences.Suitable reporter gene constructs are described in Todd, M. et al.(1993) J. Exp. Med. 177:1663-1674. See also the Examples fordescriptions of IL-4 reporter gene constructs.

A variety of reporter genes are known in the art and are suitable foruse in the screening assays of the invention. Examples of suitablereporter genes include those which encode chloramphenicolacetyltransferase, beta-galactosidase, alkaline phosphatase orluciferase. Standard methods for measuring the activity of these geneproducts are known in the art.

A variety of cell types are suitable for use as an indicator cell in thescreening assay. Preferably a cell line is used which does not normallyexpress c-Maf, such as a B cell (e.g., the M12 B lymphoma cell line) ora Th1 cell clone (e.g., AE7 cells). Nonlymphoid cell lines can also beused as indicator cells, such as the HepG2 hepatoma cell line.

In one embodiment, the level of expression of the reporter gene in theindicator cell in the presence of the test compound is higher than thelevel of expression of the reporter gene in the indicator cell in theabsence of the test compound and the test compound is identified as acompound that stimulates the expression or activity of the transcriptionfactor. In another embodiment, the level of expression of the reportergene in the indicator cell in the presence of the test compound is lowerthan the level of expression of the reporter gene in the indicator cellin the absence of the test compound and the test compound is identifiedas a compound that inhibits the expression or activity of thetranscription factor.

Alternative to the use of a reporter gene construct, compounds thatmodulate the expression or activity of c-Maf can be identified by usingother “read-outs.” For example, an indicator cell can be transfectedwith a c-Maf expression vector, incubated in the presence and in theabsence of a test compound, and Th2-associated cytokine production canbe assessed by detecting cytokine mRNA (e.g., IL-4 mRNA) in theindicator cell or cytokine secretion (i.e., IL-4 secretion) into theculture supernatant Standard methods for detecting cytokine mRNA, suchas reverse transcription-polymerase chain reaction (RT-PCR) are known inthe art. Standard methods for detecting cytokine protein in culturesupernatants, such as enzyme linked immunosorbent assays (ELISA) arealso known in the art. For further descriptions of methods for detectingcytokine mRNA and/or protein, see also the Examples.

In another embodiment, the invention provides a screening assay foridentifying proteins in Th2 cells that interact with c-Maf. These assayscan be designed based on the two-hybrid assay system (also referred toas an interaction trap assay) known in the art (see e.g., Field U.S.Pat. No. 5,283,173; Zervos et al. (1993) Cell 72:223-232; Madura et al.(1993) J. Biol. Chem. 268:12046-12054; Bartel et al. (1993)Biotechniques 14:920-924; and Iwabuchi et al. (1993) Oncogene8:1693-1696). The two-hybrid assay is generally used for identifyingproteins that interact with a particular target protein. The assayemploys gene fusions to identify proteins capable of interacting toreconstitute a functional transcriptional activator. The transcriptionalactivator consists of a DNA-binding domain and a transcriptionalactivation domain, wherein both domains are required to activatetranscription of genes downstream from a target sequence (such as anupstream activator sequence (UAS) for GAL4). DNA sequences encoding atarget “bait” protein are fused to either of these domains and a libraryof DNA sequences is fused to the other domain. “Fish” fusion proteins(generated from the fusion library) capable of binding to thetarget-fusion protein (e.g., a target GAL4-fusion “bait”) will generallybring the two domains (DNA-binding domain and transcriptional activationdomain) into close enough proximity to activate the transcription of areporter gene inserted downstream from the target sequence. Thus, the“fish” proteins can be identified by their ability to reconstitute afunctional transcriptional activator (e.g., a functional GAL4transactivator).

This general two-hybrid system can be applied to the identification ofproteins in Th2 cells that interact with c-Maf by construction of atarget c-Maf fusion protein (e.g., a c-Maf/GAL4 binding domain fusion asthe “bait”) and a cDNA library of “fish” fusion proteins (e.g., acDNA/GAL4 activation domain library), wherein the cDNA library isprepared from mRNA of Th2 cells, and introducing these constructs into ahost cell that also contains a reporter gene construct linked to aregulatory sequence responsive to c-Maf (e.g., a MARE sequence, forexample a region of the IL-4 promoter, as discussed above). cDNAsencoding proteins from Th2 cells that interact with c-Maf can beidentified based upon transactivation of the reporter gene construct.Accordingly, the invention provides a method for identifying a proteinin a Th2 cell that interacts with c-Maf comprising:

a) providing a two hybrid assay including a host cell which contains

-   -   i) a reporter gene operably linked to a transcriptional        regulatory sequence;    -   ii) a first chimeric gene which encodes a first fusion protein,        said first fusion protein including c-Maf;    -   iii) a library of second chimeric genes which encodes second        fusion proteins, the second fusion proteins including proteins        derived from Th2 cells;

wherein expression of the reporter gene is sensitive to interactionsbetween the first fusion protein, the second fusion protein and thetranscriptional regulatory sequence;

b) determining the level of expression of the reporter gene in the hostcell; and

c) identifying a protein in a Th2 cell that interacts with c-Maf.

Alternatively, a “single-hybrid” assay, such as that described inSieweke, M. H. et al. (1996) Cell 85:49-60, can be used to identifyproteins from Th2 cells that interact with c-Maf. This assay is amodification of the two-hybrid system discussed above. In this system,the “bait” is a transcription factor from which the transactivationdomain has been removed (e.g., c-Maf from which the amino-terminaltransactivation domain has been removed) and the “fish” is a non-fusioncDNA library (e.g., a cDNA library prepared from Th2 cells). Theseconstructs are introduced into host cells (e.g., yeast cells) that alsocontains a reporter gene construct linked to a regulatory sequenceresponsive to the transcription factor (e.g., a MARE sequence, forexample a region of the IL-4 promoter, responsive to c-Maf). cDNAsencoding proteins from Th2 cells that interact with c-Maf can beidentified based upon transactivation of the reporter gene construct.

In yet another embodiment, the invention provides a screening assay foridentifying compounds that modulate the interaction of c-Maf with a MAREin an IL-4 gene regulatory region. Assays are known in the art thatdetect the interaction of a DNA binding protein with a target DNAsequence (e.g., electrophoretic mobility shift assays, DNAse Ifootprinting assays and the like; for further descriptions see theExamples). By performing such assays in the presence and absence of testcompounds, these assays can be used to identify compounds that modulate(e.g., inhibit or enhance) the interaction of the DNA binding proteinwith its target DNA sequence. Accordingly, the invention provides amethod for identifying a compound that modulates the interaction of ac-Maf protein with a maf response element (MARE) of an IL-4 generegulatory region, comprising:

a) providing a c-Maf protein and a DNA fragment comprising a MARE of anIL-4 gene regulatory region;

b) incubating the c-Maf protein and DNA fragment in the presence of atest compound;

c) determining the amount of binding of the c-Maf protein to the DNAfragment in the presence of the test compound;

d) comparing the amount of binding of the c-Maf protein to the DNAfragment in the presence of the test compound with the amount of bindingof the c-Maf protein to the DNA fragment in the absence of the testcompound; and

e) identifying a compound that modulates the interaction of a c-Mafprotein with a MARE of an IL-4 gene regulatory region.

In one embodiment, the amount of binding of the c-Maf protein to the DNAfragment in the presence of the test compound is greater than the amountof binding of the c-Maf protein to the DNA fragment in the absence ofthe test compound, in which case the test compound is identified as acompound that enhances binding of c-Maf to the MARE. In anotherembodiment, the amount of binding of the c-Maf protein to the DNAfragment in the presence of the test compound is less than the amount ofbinding of the c-Maf protein to the DNA fragment in the absence of thetest compound, in which case the test compound is identified as acompound that inhibits binding of c-Maf to the MARE.

This invention is further illustrated by the following examples whichshould not be construed as limiting. The contents of all references,patents and published patent applications cited throughout thisapplication are hereby incorporated by reference. Nucleotide and aminoacid sequences deposited in public databases as referred to herein arealso hereby incorporated by reference.

EXAMPLE 1 Cytokine Specificity is Due to a Positive Transacting Factorand Not to a Repressor

Tissue specificity can be achieved through the action of repressor orsilencer proteins. Thus it was possible that the IL-2 and IL-4 geneswere actively repressed in Th2 and Th1 cells respectively. To test forthe existence of repressor proteins, somatic cell fusions were performedbetween a Th1 (D1.1) and a Th2 (D10) clone of differing MHC Class Ihaplotypes. The Th1 clone D1.1 (K^(d)) and the Th2 clone D10 (K^(k))were fused according to the “suspension cell fusion” procedure (Lane, R.D. et al. (1986) Methods Enzymol. 121:183-192). After fusion, the cellswere allowed to recover for 8 hours and then double-stained usingPE-conjugated anti-K^(k) and FITC-conjugated anti-K^(d) antibodies(Pharmingen, La Jolla, Calif.). Cells were then sorted on the basis ofsize to distinguish unfused cells from hetero and homokaryons and byfluorescence to identify single-positive and double-positive cells. Asindicated in the schematic of this approach shown in FIG. 1A, threepopulations were sorted for: large PE-positive cells (D1.1×D1.1), largeFITC-positive cells (D10×D10), and large PE and FITC positive cells(D1.1×D10). Cells expressing both MHC class I K^(b) and K^(k) markerswere heterokaryons while cells expressing only K^(b) or K^(k)represented homokaryons and served as controls.

The three populations were then stimulated in culture with antibodies toCD3 to activate cytokine gene expression and RNA prepared for RT-PCR andNorthern blot analysis. Approximately 5×10⁵ cells were obtained for eachpopulation. Routinely, 5-10% of the cells had undergone fusion. Each ofthese three populations was then split in half, one half transferred topre-rinsed anti-CD3 coated plates, the remaining half to uncoatedplates. After four hours, the cells were harvested, and poly(A+) RNAisolated using the Micro-FastTrack™ kit (Stratagene, La Jolla, Calif.).cDNA was made using the SuperScript kit (Gibco/BRL, Bethesda, Md.), andused for PCR analysis using commercially available primers specific formurine IL-2, IL-4 and β-actin according to the manufacturer'sinstructions (Stratagene, La Jolla, Calif.). PCR reactions included 0.5μCi α-³²P-dCTP (3000 Ci/mmol, NEN Dupont). PCR products were ethanolprecipitated, separated by nondenaturing PAGE and dried and visualizedby autoradiography.

The results of the RT-PCT analysis of cytokine mRNA expression are shownin FIG. 1B. The Th1 and Th2 clones and the Th homokaryons transcribedonly IL-2 (Th1) or IL-4 (Th2) respectively, while the Th1/Th2heterokaryons produced both cytokines. In contrast, the existence ofrepressor protein(s) should have resulted in the extinction of bothcytokines in the heterokaryons. From these experiments, it was concludedthat cytokine specificity in Th1 vs. Th2 cells was mediated byTh-specific positive transacting factors rather than by selectivesilencer proteins.

EXAMPLE 2 Isolation of a Th2-Specific c-maf Gene from a cDNA LibraryPrepared from an Anti-CD3 Activated Th2 Clone

In the course of screening a cDNA library prepared from an anti-CD3activated Th2 clone, D10, for NF-AT-interacting proteins by the yeasttwo-hybrid system (for descriptions of this system, see e.g., Field U.S.Pat. No. 5,283,173; Zervos et al. (1993) Cell 72:223-232; Madura et al.(1993) J. Biol. Chem. 268:12046-12054; Bartel et al. (1993)Biotechniques 14:920-924; and Iwabuchi et al. (1993) Oncogene8:1693-1696), multiple cDNAs were isolated, all of which were extremelyweak interactors. All cDNAs obtained in this screen were next evaluatedfor Th-specific expression by Northern blot analysis using a panel ofTh1 and Th2 clones. One such cDNA, which was repeatedly isolated (60 of140) detected transcripts only in RNA prepared from Th2 clones (D10,CDC35) and not from either Th1 clones (AR5, OS6, D1) or from a B celllymphoma, M12, as illustrated in the Northern blot analysis depicted inFIG. 2A. Further, the levels of transcripts detected in D10 Th2 cellswere substantially increased upon activation by ligation of the T cellreceptor with anti-CD3 antibody. No induction of the transcript detectedby this cDNA clone occurred in Th1 clones upon anti-CD3 treatment. Acontrol probe, GAPDH, demonstrated approximately equal loading of RNA inall lanes. Thus, the expression of this cDNA clone in the lymphoidlineage appeared to be Th2-specific and sensitive to signals transmittedthrough the T cell receptor. For these Northern blots, total RNA wasprepared by using Trizol (GIBCO/BRL) according to manufacturer'sinstructions. 10 μg of total RNA from each sample was fractionated on aformaldehyde agarose gel and transferred to a nylon membrane. A 300 bpDraI fragment derived from the 3′ untranslated region of the isolatedclone was labeled with α-³²P-dCTP using Random Primed DNA Labeling Kit(Boehringer Mannheim, Indianapolis, Ind.). Hybridization was performedusing QuikHyb (Stratagene, La Jolla, Calif.) according to manufacturer'sinstructions.

To determine whether the expression of this gene was tissue-specific andregulated during the course of normal Th cell development, the followingexperiment was performed. Naive spleen cells (Th precursor (Thp) cells)were driven along a Th1 or Th2 pathway by treatment with anti-CD3 in thepresence of cytokines and anti-cytokine antibodies (IFNγ and anti-IL-4for Th1, IL-4 and anti-IFNg for Th2). Splenic cell suspensions wereprepared from 6-8 week-old Balb/c mice, cultured in RPMI 1640supplemented with 10% FCS at a density of 10⁶ cells/ml, and stimulatedwith plate bound anti-CD3 antibody in the presence of 5 μg/ml ofanti-IL4 antibody (11B11) for the Th1 lineage, or 5 μg/ml of anti-IFNγantibody (XMG-1) for the Th2 lineage. 24 hours after stimulation, 50U/ml IL-2 was added to all cultures, and 500 U/ml IL-4 (Genzyme) wasadded to Th2 cultures. 7 days after the primary stimulation, all cellswere harvested, washed and restimulated with plate bound anti-CD3antibody. Northern blot analysis of differentiating cells harvested atvarious time points after stimulation in a primary (day 0-8) andsecondary (0-20 hours) response was performed, using the methodologydescribed above, and identification of differentiating Thp cells as Th1or Th2 was determined by analyzing culture supernatants by ELISA forIL-10 and IFNγ. ELISA for cytokine quantitation was performed asfollows. All anti-cytokine antibodies were purchased from Pharmingen.ELISA was performed according to Pharmingen's instructions with theexception that Avidin-Alkaline Phosphatase (Sigma) at 1:500 dilution inPBS/BSA was used in place of avidin-peroxidase. P-nitrophenyl phosphate(GIBCO BRL) at 4 mg/ml in substrate buffer (10% diethanolamine, 0.5 mMMgCl₂, 0.02% sodium azide, pH 9.8) was used as substrate.

In two independent experiments, representative results of which areshown in FIG. 2B, this analysis revealed low level or undetectableexpression of this cDNA in naive spleen cells at baseline at day 0. Incultures differentiating along a Th2 pathway, substantial induction oftranscripts occurred by day 8 in a primary stimulation and by 20 hr in asecondary stimulation. In contrast, no induction occurred in cells beingdriven along a Th1 pathway. A control probe (GAPDH) showed approximatelyequal loading of RNA in all lanes. The low level of transcripts presentin cells being driven along a Th1 pathway likely reflects the presenceof residual Th2 cells since complete skewing does not occur in this invitro differentiation system.

Together, these experiments revealed that the isolated cDNA isselectively expressed in Th2 clones, where it is induced upon T cellactivation, and that it is absent from Th1 clones and a B lymphoma.Further, this gene is induced in normal Thp when they are driven towardsthe Th2 lineage, but is not induced during Th1 development.

The cDNA obtained from the yeast two-hybrid screen was used as a probeto isolate a full-length cDNA from a D10 Th2 cell cDNA library bystandard hybridization methods. A 4.3 kb cDNA clone was isolated fromthe Th2 cell library and sequenced by standard methods. Sequenceanalysis revealed that this Th2-specific gene corresponded in sequenceto the c-maf proto-oncogene.

EXAMPLE 3 Ectopic Expression of c-Maf in Th1 and B Cells Results inActivation of the IL-4 Promoter

The identification of the isolated cDNA described in Example 2 as amember of the AP-1/CREB/ATF gene family, together with its selectiveexpression in Th2 cells raised the possibility that c-Maf controlled thetissue-specific transcription of the IL-4 gene. Additionally, thepresence of transcripts encoding c-maf correlated well with IL-4expression in Th2 cells and in three of four transformed mast cell linesexamined. To test whether c-Maf could transactivate the IL-4 promoter,cotransfection experiments were performed.

Th1 clones and the B lymphoma M12.4.C3 (M12) neither express c-maf nortranscribe the IL-4 gene. If c-Maf is the transcription factor criticalfor controlling IL-4 gene expression, then forced expression in thesecells should permit IL-4 gene expression. To test this, the full-length(4.3 kb) c-maf cDNA clone was inserted into the SalI site of thepMex-NeoI mammalian expression vector, which utilizes the CMV enhancerto drive expression of the inserted sequence. The c-Maf expressionvector was then cotransfected with an IL-4 promoter reporter constructinto the Th1 clone AE7 and the B lymphoma M12. The generation of thewild type IL4 CAT reporter construct, containing an IL-4 promoterfragment from −157 to +68 operatively linked to a chloramphenicolacetyltransferase gene is described in Hodge, M. et al. (1995) J.Immunol. 154:6397-6405. The Th1 clone was cultured in RPMI 1640supplemented with 10% FCS and 10% Con-A stimulated rat splenocytesupernatant, and maintained by bi-weekly stimulation with appropriateantigen and APCs. M12 cells were cultured in RPMI 1640 supplemented with10% FCS.

The Th1 clone AE7 or M12 B lymphoma cells were transiently transfectedby preincubating 0.4 ml of cells, containing 2×10⁷ cells/ml AE7 or 3×10⁶cells/ml M12 cells, in serum-free RPMI 1640 with 20 μg (AE7) or 5 μg(M12) of each plasmid for 10 minutes at room temperature. The sampleswere then electroporated using a BIO-RAD Gene Pulser (BIO-RAD, Richmond,Calif.) set at 975 μF, 280 V, and immediately placed on ice for 10minutes. The transfected cells were allowed to recover overnight incomplete media and stimulated with plate bound anti-CD3 antibody({Pharmingen, San Diego, Calif.} 1 μg/ml in 1×PBS overnight at 4° C.) orwith 50 ng/ml PMA (Sigma, St. Louis, Mo.) and 1 μM Ionomycin (CalbiochemCorp., La Jolla, Calif.). for 24 hours. Cell lysate was prepared byfreeze-thaw lysis in 0.25 M Tris-Cl, pH 7.8. Equal amounts of protein(between 5-20 μg) were used for CAT assays. CAT assays were performed asdescribed in Todd, M. et al. (1993) J. Exp. Med. 177:1663-1674.

It has previously shown that Th2-specific, inducible IL-4 expression canbe directed by as little as 157 bp of the proximal IL-4 promoter in Th2cells (Hodge, M. et al. (1995) J. Immunol. 154:6397-6405). Incotransfection experiments, the results of which are summarized in FIG.3A, it is demonstrated that ectopic expression of c-Maf in the Th1 cloneAE7 results in substantial activity of the IL-4 promoter reporter afterstimulation through the T cell receptor. The fold induction observed wasapproximately 5 fold over that observed with the control empty vectoralone. Although expression of a reporter construct containing proximal(−157 to +58) IL-4 promoter sequences in the subclone of AE7 cellsutilized here has not been previously observed, it has been demonstratedthat small amounts of IL-4 mRNA can be detected by RT-PCR in othersubclones of AE7. To more rigorously test the ability of c-Maf totransactivate the IL-4 promoter in a non-IL-4 producing cell, the sameexperiment was performed in the B lymphoma cell line, M12. Normal Bcells and B lymphoma cells do not produce IL-4. Representative resultsof the cotransfection experiments are depicted in FIG. 3B and a summaryof three independent experiments is shown below in Table 1.

TABLE 1 CAT Activity (fold induction) Plasmids PMA/iono. Exp. I* Exp. IIExp. III pMEX-NeoI/pREP4 − 1 1 1 + 7.6 1 1.4 pMEX-Maf/pREP4 − 95 518.6 + 186 7 37 pMEX-c-Fos/pREP4 − 2.7 1 0.8 + 7.6 1.2 1 pMEX-JunD/pREP4− ND** 0.9 0.5 + ND 1.4 1.9 pMEX-NeoI/pREP4-NFATp − 14.2 1.6 0.3 + 41.23.5 0.3 pMEX-Maf/pREP4-NFATp − 136 54 26.3 + 138 100 54.7pMEX-c-Fos/pREP4-NFATp − 7.4 1.6 3 + 15.4 1.9 6.1 *In experiment I, 20mg of cell lysate was incubated for 2 hours. In experiments II and III,only 5 mg of cell lysate was incubated for 1 hour in order to revealsynergy between c-Maf and NFATp **ND = not done

The results in M12 B lymphoma cells confirmed the findings in the Th1clone. Ectopic expression of c-Maf resulted in substantial activity ofthe IL-4 promoter in M12 cells, either unstimulated or stimulated withPMA/Ca++ ionophore. The fold induction observed when compared totransfection of a control vector averaged approximately 50 inunstimulated M12 cells. Stimulation of M12 cells with PMA/Ca++ionophore, which should result in translocation of NF-ATs to the nucleusand induction of other AP-1 family members (Flanagan, W. M. (1991)Nature 352:803-807; Jain, J. et al. (1993) Nature 3:352-355), increasedthe basal activity of the IL-4 promoter, but a marked induction inpromoter activity by c-Maf was still present (average of approximately25 fold). C-Maf did not transactivate a control reporter driven by NF-ATmultimers, demonstrating the specificity of transactivation.

As a control for the specificity of c-Maf as opposed to other AP-1family members, the c-Fos and c-Jun proteins were also overexpressed inM12 cells utilizing murine full-length cDNAs encoding c-Fos and JunD inthe mammalian expression vector of pMEX-NeoI together with the IL-4reporter plasmid. No IL-4 promoter activity could be achieved byoverexpression of either of these two AP-1 family members in M12 cells.Thus, c-Maf has a unique ability to drive IL-4 gene transcription in M12B cells. Further, forced expression of c-Maf in the hepatoma cell lineHepG2 also resulted in IL-4 promoter transactivation. These experimentsdemonstrate that the provision of c-Maf to c-Maf negative Th1 or Bcells, or to non-lymphoid cells (e.g., a hepatoma cell line), permitsthe cells to transactivate the IL-4 promoter.

NF-AT proteins have been shown to be critically important in theregulation of both the IL-4 and IL-2 cytokines. NF-ATp was the firstmember of this family to be isolated (McCaffrey, P. G. et al. (1993)Science 262:750-754). Both AE7 and M12 cells have endogenous NF-ATpprotein, but nevertheless do not transcribe IL-4. Although NF-ATp couldnot therefore account for selective IL-4 gene transcription, it was ofinterest to test whether overexpression of NF-ATp in unstimulated orstimulated M12 cells would further increase the transactivation of theIL-4 promoter by c-maf. M12 cells were cotransfected with the IL-4reporter construct and either an NFAPp expression vector (pREP₄-NF-ATp,which also carries a hygromycin resistance gene) alone or the NFAPpexpression vector together with the c-Maf expression vector.Overexpression of NF-ATp alone in M12 cells resulted in some modesttransactivation of the IL-4 promoter. This transactivation was markedlyincreased by ectopic expression of c-Maf, an increase which was not justadditive but was synergistic (see FIG. 3B and Table 1). In contrast,c-Fos overexpression did not further increase the modest transactivationachieved by NF-ATp. These results indicate that c-maf and NF-ATpinteract to achieve maximal induction of the IL-4 promoter, thetissue-specificity being provided by c-maf.

EXAMPLE 4 Ectopic Expression of C-Maf Activates Transcription of theEndogenous IL-4 Gene in a B Lymphoma

As demonstrated in Example 3, c-Maf transactivates the IL-4 promoter intransient transfection assays in Th1, B and non-lymphoid cells. To testwhether expression of c-maf in non-IL-4 producing cells can activate thetranscription of endogenous IL-4, the B lymphoma M12 was stablytransfected with expression vectors encoding c-maf, NF-ATp or both, orjunD with and without NF-ATp as a control. For stable transfection, M12cells were transfected as described above in Example 3. The transfectedcells were allowed to recover in complete media for 48 hours before theaddition of Neomycin (GIBCO/BRL, Gaithersburg, Md.) and Hygromycin(Calbiochem, Corp.) at a concentration of 400 μg/ml of each antibiotic.The transfected cells were supplemented with fresh media every otherday.

Stably transfected M12 cells were plated at equal density supernatantsharvested 24 hours later to measure cytokines by ELISA. ELISAs wereperformed as described in Example 2. The results, shown in FIG. 4,demonstrate that in these experiments M12cells transfected with c-maf,junD or NF-ATp alone did not produce measurable IL-4 by ELISA. However,M12 cells stably transfected with both c-maf and NF-ATp did producedetectable, but low level, IL-4 by ELISA. These results were confirmedby RT-PCR on RNA from these transfected cells. In contrast, these cellsdid not produce detectable IL-2. The requirement for both c-maf andNF-ATp is consistent with the synergistic effect of these factors in thetransactivation of the IL-4 promoter noted in the transient transfectionexperiments in M12 cells. In contrast, transfection of junD, an AP-1family member which can increase IL-4 expression in Th2 cells, alone ortogether with NF-ATp, did not result in IL-4 production. These resultsdemonstrate the essential and selective role of c-maf in directingtissue-specific endogenous IL-4 production.

EXAMPLE 5 A Site in the IL-4 Promoter is Footprinted by Extracts fromTh2 but not Th1 Clones

The experiments described in Examples 3 and 4 demonstrated a clearfunctional role for c-maf in controlling tissue-specific expression ofIL-4. Further, c-maf transcripts were expressed in Th2 but not Th1cells. However, DNA-protein complexes were not detected byelectrophoretic mobility shift assays (EMSA) when using nuclear extractsprepared from Th2 cells. To further examine whether a protein in Th2nuclear extracts might bind to the MARE, or nearby sequences, the moresensitive technique of DNAseI footprinting was used. Two Th2 clones(D10, CDC35) and two Th1 clones (AE7, S53) were activated by ligation ofthe T cell receptor with plate-bound anti-CD3 antibody, and nuclearextracts prepared at time 0 (unstimulated), 2 hours and 6 hours later.DNAseI footprinting analysis was then performed according to standardmethods using a Klenow end-labeled IL-4 promoter fragment (−157 to +68).The results are shown in FIG. 5A. Stimulated extracts from both Th1 andTh2 cells footprinted the two NF-AT sites and the AP-1 site upstream ofthe distal NF-AT site as described previously (Rooney, J. et al. (1995)Immunity 2:545-553), consistent with the demonstrated function of NF-ATand AP-1 proteins in regulating both the IL-2 and the IL-4 promoters(Rooney, J. et al. (1995) Immunity 2:545-553; Rooney, J. et al. (1995)Mol. Cell. Biol. 15:6299-6310). Furthermore, inspection of theautoradiograph revealed an area of hypersensitivity on the non-codingstrand at residues −28 and −29 when extracts from stimulated Th2 but notstimulated Th1 cells were used. Unstimulated Th cell extracts did notfootprint this region. The Th2 footprint observed was subtle, butreproducible in two experiments and is located in a site that haspreviously been demonstrated to be critical for IL-4 promoter activationin Th2 cells (Hodge, M. et al. (1995) J. Immunol. 154:6397-6405). Aschematic summary of sites occupied in the IL-4 promoter as detected byfootprint analysis is shown in FIG. 5B. These results indicate that asite in the proximal IL-4 promoter, previously shown to be functionallyimportant, is occupied in activated Th2 but not in activated Th1 cells.

EXAMPLE 6 Recombinant c-maf Binds to a MARE Site in the IL-4 Promoter

The Th2-specific footprint does not contain a c-maf response element(MARE). However, examination of the proximal IL-4 promoter revealed ahalf c-maf binding site (MARE) (residues −42 to −37) immediatelydownstream of the proximal NF-AT site (residues −56 to −51) (shownschematically in FIG. 5B). It has previously been demonstrated thatmutation of this site abolished activity of the IL-4 promoter in Th2cells (Hodge, M. et al. (1995) J. Immunol. 154:6397-6405). To determineif c-Maf bound this site, a truncated c-Maf recombinant proteincontaining the b-zip domain(amino acids 171-371) was expressed from E.coli, purified on an S-Tag agarose column and used in electrophoreticmobility shift assays with radiolabeled MARE oligonucleotide.

The expression vector for recombinant c-Maf was constructed by insertinga cDNA fragment encoding a.a. residues 171 to 371 of c-Maf (disclosed inKurschner C. and Morgan, J. I. (1995) Mol. Cell. Biol. 15:246-254) intothe NotI site of pET29 (Novagen, Inc. Madison, Wis.). The truncatedc-Maf protein was expressed using T7 polymerase in the BL21(DE3)strain.Cells were induced by the addition of 1 mM IPTG and incubated at 37° C.for 3 hours. The induced cells were lysed in 1× Bind/Wash buffer (20 mMTris-HCl pH 7.5, 150 mM NaCl, 0.1% Triton X-100) followed by sonication.The c-Maf protein was then purified from the soluble fraction by usingthe S-Tag Purification Kit (Novagen) according to manufacturer'sinstructions. Two additional proteins, NF-ATp and c-Jun, were also usedin EMSA assays. The recombinant NF-ATp, containing the Rel domain ofmurine NF-ATp, was expressed using an in vitro transcription/translationvector TP7-NF-ATp, which contains a cDNA fragment encoding the Reldomain of murine NF-ATp. The c-Jun expression vector, pGEM-c-Jun, wasconstructed by inserting a full-length cDNA of murine c-Jun into thePstI site of pGEM4. 1 μg of each plasmid DNA was transcribed from the T7promoter and translated in rabbit reticulocyte lysate by using the TnTCoupled Transcription/Translation Kit (Promega, Madison, Wis.).

Electrophoretic mobility shift assays (EMSA) were performed as follows.100 ng of double-stranded oligonucleotides were end-labeled withγ-³²P-dATP (DuPont NEN Research Product, Wilmington, Del.) using T4polynucleotide kinase (Pharmacia LKB Biotechnology, Inc., Piscataway,N.J.). The labeled ds-oligonucleotides were fractionated on 15-20%polyacrylamide gels, eluted overnight at 37° C. in 1×TE and precipitatedin ethanol. Binding assays were performed at room temperature for 20minutes using 0.5 μg of recombinant proteins or 4 μl of in vitrotranslated products, 500 ng poly(dI-dC), and 20,000 cpm of probe in a 15μl volume of 20 mM HEPES (pH 7.9), 100 mM KCl, 5% glycerol, 1 mM EDTA, 5mM DTT, 0.1% NP-40, and 0.5 mg/ml BSA. The samples were thenfractionated in 4% non-denaturing polyacrylamide gel containing 0.5×TBEat room temperature.

Oligonucleotides derived from the murine IL-4 promoter used in EMSAwere:

-   −59 to −27: 5′-CTCATTTTCCCTTGGTTTCAGCAACTTTAACTC-3′ (SEQ ID NO: 1);-   −79 to −60: 5′-ATAAAATTTTCCAATGTAAA-3′ (SEQ ID NO:2); and-   −88 to −61: 5′-TGGTGTAATAAAATTTTCCAATGTAAA-3′ (SEQ ID NO: 3).    The sequence of the MARE oligonucleotide used in EMSA was:-   5′-GGAATTGCTGACTCAGCATTACT-3′ (SEQ ID NO: 4).    All oligonucleotides were annealed with their respective    reverse-complementary strands to form double-stranded    oligonucleotides.

The results of EMSA with recombinant c-Maf are shown in FIG. 6. Therecombinant c-Maf protein bound well to both a consensus MAREoligonucleotide and to a 33 bp oligonucleotide containing the NF-AT siteand MARE present in the IL-4 promoter. Binding was specifically competedby unlabeled homologous but not control probe. Further, c-Maf did notbind to an oligonucleotide containing only the NF-AT target sequence towhich recombinant NF-ATp bound well. The ability of c-Maf to bind to theIL-4 promoter probe was specific since in vitro translated c-Jun proteindid not bind to this oligonucleotide. The c-Jun protein was functionalsince it could bind to the consensus MARE which contains a core TREsite. These results indicate that c-Maf, but not another AP-1 familymember (c-Jun), can bind to the MARE site within the proximal IL-4promoter.

NF-AT proteins interact cooperatively with AP-1 family member proteinsto form higher mobility complexes on IL-2 and IL-4 promoter DNA on EMSA(Jain, J. (1993) Nature 365:353-355; Rooney, J. et al. (1995) Immunity2:545-553). That NF-AT proteins might interact with c-maf was suggestedby the functional studies described in the previous examples. Todetermine if c-Maf interacted with NF-AT in the presence of DNA,recombinant NF-ATp and c-Maf proteins were used separately or togetherin EMSA with the 33 bp oligonucleotide containing both the NF-AT andadjacent MARE sites. The results are shown in FIG. 6. Each protein alonebound to IL-4 promoter DNA. Recombinant c-Maf plus recombinant NF-ATpprotein produced these complexes and in addition formed a highermobility complex. No higher mobility complex was observed when c-Jun andNF-ATp proteins were used, consistent with the failure of c-Jun to bindthis site. These results indicate that c-Maf can specifically bind invitro to a sequence located in the proximal IL-4 promoter, previouslyshown to be functionally critical in Th2 cells, and that, like otherAP-1 proteins, c-Maf can interact in vitro with NF-AT proteins.

EXAMPLE 7 The Ability of c-Maf to Transactivate the IL-4 Promoter Mapsto the MARE and Th-2 Specific Footprint

An essential region of the IL-4 promoter located immediately upstream ofthe TATA element has been characterized by high resolution mutagenesis(Hodge, M. et al. (1995) J. Immunol. 154:6397-6405). Mutagenesis of this33 bp region (−59 to −28) demonstrated multiple sites required forinducible IL-4 transcription in Th2 cells. These sites included an NF-ATtarget sequence, the region footprinted by Th2 extracts, and what is nowrecognized as a MARE. A series of IL-4 reporter gene constructscomprising 4 base pair linker-scanning mutants generated across thisregion were used to map the target sequence utilized by c-Maf in vivo inM12 cells. These cells were cotransfected with the c-maf expressionvector and this series of mutant IL-4 promoter constructs. The resultsare shown in FIG. 7A. Mutation of the MARE (muts 3 and 4), or the sitedefined by the Th2 footprint (mut 2), abrogated (muts 2 and 4) orpartially abrogated (mut 3) the ability of transfected c-maf to driveIL-4 transcription. A modest effect in reducing c-maf transactivationwas also observed for mutant 8 which disrupts the NF-AT sequence,consistent with the presence in M12 cells of endogenous NF-ATp and withthe synergy between NF-ATp and c-maf demonstrated in the previousexamples. Mutants 6 and 7 had no significant effect while mutant 5 badenhanced transactivation ability, consistent with previous observationsin Th2 cells (Hodge, M. et al. (1995) J. Immunol. 154:6397-6405). Thetransactivation data is consistent with EMSA performed with recombinantc-Maf protein using as probe an oligonucleotide which contains this 33bp region, and this same series of mutant oligonucleotides as coldcompetitors. The results of these EMSA experiments are shown in FIG. 7B.These experiments indicate that c-Maf specifically binds to andtransactivates the MARE in the proximal IL-4 promoter and that theadjacent Th2-specific element is intimately involved in both the bindingand function of c-Maf.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. A method for identifying a compound that modulates production of IL-4in a cell, comprising providing an indicator composition comprising (i)a c-maf or v-maf protein and (ii) a target DNA comprising a regulatorysequence of an IL-4 gene which includes a c-maf responsive element(MARE), wherein said indicator composition is an indicator cell oracellular preparation; contacting the indicator composition with eachmember of a library of test compounds; selecting from the library oftest compounds a compound of interest that modulates binding of said maffamily protein to said target DNA; and determining the effect of thecompound of interest on the production of IL-4 in a cell to therebyidentify a compound that modulates production of IL-4.
 2. The method ofclaim 1, wherein the maf family protein is c-Maf.
 3. The method of claim1, wherein the effect of the compound of interest on IL-4 production isdetermined by determining the effect of the compound on development of Thelper type 1 (Th1) or T helper type (Th2) cells.
 4. The method of claim1, wherein the maf family protein is v-maf.
 5. The method of claim 1,wherein the indicator composition is an indicator cell.
 6. The method ofclaim 5, wherein the indicator cell is a lymphoid cell.
 7. The method ofclaim 6, wherein the lymphoid cell is a Th2 cell.
 8. The method of claim6, wherein the lymphoid cell is a Th1 cell.
 9. The method of claim 6,wherein the lymphoid cell is a B cell.
 10. The method of claim 5,wherein the indicator cell is a non-lymphoid mammalian cell.
 11. Themethod of claim 5, wherein the indicator cell is a yeast cell.
 12. Themethod of claim 6, wherein the lymphoid cell is a helper precursor (Thp)cell.
 13. The method of claim 1, wherein the indicator composition is anacellular preparation.
 14. The method of claim 1, wherein the mafprotein is recombinantly expressed in a cell.
 15. The method of claim 1,wherein the cell does not naturally express the maf protein.
 16. Themethod of claim 1, wherein the regulatory sequence comprises about 3 kbof the upstream regulatory sequences of the IL-4 gene.
 17. The method ofclaim 1, wherein the regulatory sequence comprises from about nucleotide−157 to about nucleotide +58 relative to the start site of transcriptionof +1 of the IL-4 promoter.
 18. The method of claim 1 whereinTh2-associated cytokine production is assessed by detecting IL-4 mRNA.19. The method of claim 1, wherein Th2-associated cytokine production isassessed by detecting the IL-4 protein.
 20. The method of claim 1,wherein the regulatory sequence comprises from about nucleotide −42 toabout nucleotide −37 relative to the start site of transcription of +1of the IL-4 promoter.
 21. The method of claim 1, wherein the regulatorysequence comprises from about nucleotide −59 to about nucleotide −27relative to the start site of transcription of +1 of the IL-4 promoter.